Int. J. Mol. Sci. 2015, 16, 17193-17230; doi:10.

3390/ijms160817193
OPEN ACCESS
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Review

Protein Folding and Mechanisms of Proteostasis

José Fernando Díaz-Villanueva, Raúl Díaz-Molina and Victor García-González *

Facultad de Medicina, Universidad Autónoma de Baja California, Mexicali, Baja California 21000,
México; E-Mails: [email protected] (J.F.D.-V.); [email protected] (R.D.-M.)

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +52-68-6557-1622 (ext.121); Fax: +52-68-6557-1622 (ext.109).

Academic Editor: Salvador Ventura

Received: 1 April 2015 / Accepted: 11 June 2015 / Published: 28 July 2015

Abstract: Highly sophisticated mechanisms that modulate protein structure and function,
which involve synthesis and degradation, have evolved to maintain cellular homeostasis.
Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious
cell processes. Therefore in recent years the etiology of a great number of diseases has
been attributed to failures in mechanisms that modulate protein structure. Interconnections
among metabolic and cell signaling pathways are critical for homeostasis to converge on
mechanisms associated with protein folding as well as for the preservation of the native
structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead
to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive
unfolded protein response (UPR). Therefore, taking this into consideration, a key part of
this paper is developed around the protein folding phenomenon, and cellular mechanisms
which support this pivotal condition. We provide an overview of chaperone protein
function, UPR via, spatial compartmentalization of protein folding, proteasome role,
autophagy, as well as the intertwining between these processes. Several diseases are known
to have a molecular etiology in the malfunction of mechanisms responsible for protein
folding and in the shielding of native structure, phenomena which ultimately lead to
misfolded protein accumulation. This review centers on our current knowledge about pathways
that modulate protein folding, and cell responses involved in protein homeostasis.

Keywords: proteins; folding; proteostasis; misfolding

Int. J. Mol. Sci. 2015, 16 17194

1. Introduction

One of the keystones that has been considered to drive the evolution of organisms, relies on the
capacity to detect, respond, and adapt to various stressors in the environment through cellular defensive
mechanisms that protect the entire organism and maintain its capacity to grow and reproduce [1].
For instance, establishment of preformed enzyme complexes for cell function has been proposed as a
mechanism used by cells to rapidly respond to homeostasis unbalances [2,3], whereas pathways that
regulate the conservation of protein folding have a key role [4].
Function in proteins largely depends on the acquisition of specific three-dimensional structures by
folding at physiological time scales. Cells have developed highly controlled mechanisms to maintain
native protein folding, which include detail three-dimensional structure patterns and specific disordered
domains. Notwithstanding, although the process is extremely efficient, there is always the possibility that
this accurate mechanism fails, and, in consequence, finding a protein folded into a non-native state
becomes a reality [5]. In this case, the structural stability of proteins depends largely on hydrophobic
residues being oriented towards the protein core. This has allowed evolution to develop a conserved
warning system, in which the exposure of protein hydrophobic regions is recognized as a molecular
pattern associated with the presence of cytotoxicity [6]. For instance, oligomerization preceding
amyloid fibril formation has been associated with cytotoxic effects, which may arise from their
misfolded conformation in which hydrophobic side chains are exposed to the cytosol [7,8].
Cytotoxic effects could be triggered by interactions among hydrophobic regions of proteins exposed to
aqueous microenvironment with cellular biomolecules, such as other proteins, nucleic acids, or lipid
membranes [9,10].
Cells modulate protein folding and protein degradation through extensive signaling networks to
avoid misfolded protein accumulation [11]. Likewise, cells must not only promote accurate protein
folding but also prevent the accumulation of misfolded species that may arise from translation errors,
and synthesis of aberrants mRNAs [12]. Proteomes have been described as multifaceted and constantly
evolving entities [13]. Then, the understanding of the quality control checkpoints, which are performed
during normal cell physiology, and those that are activated during protein stress, including the stress
responses, function of catabolic machineries, and systems of communication among molecules and
organelles, are subject to an extensive research.
Most of the signaling proteins used by eukaryotic cells to communicate with their environment are
assembled in endoplasmic reticulum (ER). Transmission and management of information through
proteins is crucial for the homeostasis of the organisms, regulating mechanisms such as cell cycle,
apoptosis, and cell growth [14]. Loss of protein structure can arise from alterations in diverse stages
during protein synthesis, the degradation process, or changes in concentration of metabolites in the cell
environment. Given the central role of protein folding in biology, it is interesting to think that a
misfolding can lead to dysfunction, modifying the cellular mechanism in which the protein is involved.
In fact, many chronic-degenerative diseases are associated with an aberrant protein folding, modifying
the condition of protein homeostasis (proteostasis) to cause situations such as ER stress [15–17].
Several points of interconnection within pathways responsible for maintaining the cell functions
converge on mechanisms of protein folding, and therefore, on the conservation of native three
dimensional structure of proteins. In this paper, the main focus will rely on the analyses of mechanisms
Int. J. Mol. Sci. 2015, 16 17195

related with the chaperone protein function and unfolded protein response (UPR) pathways, in addition
to strategies developed by cells such as the spatial compartmentalization of protein folding, protein
degradation by proteasomes, and autophagy; all of these mechanisms connected with the objective to
conserve the proteostasis (Figure 1). Misfolding condition seen through various topics is a fundamental
field in the understanding of protein nature, and cells devote many resources for its regulation.
Therefore, the understanding of these molecular mechanisms is a cornerstone for optimum
pharmacological treatments of diseases associated with protein misfolding.

Figure 1. Molecular and cellular mechanisms to maintain native protein structure. The
vertical arrows indicate the mechanisms which cells employ to counteract alterations in
native protein structure, during their lifetime (horizontal arrow). When the cellular
machinery designed to control widespread protein misfolding or aggregation fails, apoptosis
ensues. When apoptosis cannot restrict the systemic spread of protein misfolding, chronic
diseases originate (red arrow).

2. Protein Structure

2.1. Protein Folding

Cell functions need to be regulated with high levels of efficiency, a condition under strict control is
the acquisition and maintenance of the native three-dimensional structure of proteins. A protein
acquires its functional structure through a folding phenomenon, in which its amino acid sequence
acquires the minimum energy conformation [18]. For folding into a native state, unfolded polypeptide
chains require the intervention of weak interactions. Driven by hydrophobic interactions, a polypeptide
chain begins to fold when it is present in an aqueous environment after synthesis, and rapidly becomes
a molten globule followed by an important release of latent heat. Stabilization of the molten globule is
achieved mainly through the distribution of hydrophobic residues away from the bulk water. On the
other hand, because the polar residues contained in a protein develop hydrogen bonds with the water
network as well as with each other, α-helices and β-sheets can be formed when bonds switch between
molecules. It has been calculated that such bonds might be in the order of 10−12 s, very similar to those
we find in water itself [19].
The random equilibrium can be shifted toward one of these conformations by means of two stages:
a fast stage, during which the unfolded polypeptide becomes a molten globule; and a slow stage, in
Int. J. Mol. Sci. 2015, 16 17196

which the molten globule slowly transforms into a fully folded native state [19]. Considering that the
native state is located at the minimum of the folding funnel, it indicates that this region is the most
thermodynamically stable configuration of the polypeptide chain [20].
Chains of amino acids must acquire their three-dimensional structures in very short time scales,
which is a requirement to operate within a highly concentrated cellular environment. Although varied
and intricate structures of globular proteins are encoded by their amino acid sequences, and these
molecules have an intrinsic ability to fold spontaneously [21], under normal physiological conditions,
some proteins do not fold into globular structures [22,23], and a negative effect on cell physiology
might occur.

2.2. Allosteric Approach to Proteins

Proteins have been visualized as dynamical objects that interconvert between a diversity of
structures with varying energies, rather than static sculptures [24]. In this sense, these biomolecules can
be physicochemically described not as single structures but, rather as conformational ensembles with
dynamic distributions of states, which can change under different micro-environmental conditions [25].
This feature represents a possible association with a new base to understand the allostery, a phenomenon
related in the first case to describe the dynamic modularity of proteins, and subsequently in the
possible understanding of cellular dynamics.
While the function of several proteins has been characterized from an in vitro approach, information
regarding the influence of the highly crowded cellular environment, or the effect of metabolites
and molecules on protein function is not complete. Membrane systems, multi-enzyme complexes,
macromolecular structures, and metabolite concentrations should have a direct influence on protein
function and, ultimately in the regulation of cell physiology [26]. For example, perturbations in
the native three-dimensional structure of proteins does not produce isolated effects on the single
protein but, rather could affect the assembly of macromolecular structures, modifying the surrounding
microenvironment [27].
Traditionally, the allosteric phenomenon has been associated with conformational and functional
transitions on individual proteins; for instance, regulating many features of enzyme performance.
However, some authors have extended this concept to include the impact of protein conformational
perturbations on cellular function [25,28]. It has been proposed that multi-scale organization across
different levels provides a feedback regulation on specific proteins, and collectively on cell signaling
pathways. Through this perspective, proteins perform their functions by highly interconnected cellular
pathway linkages. Therefore, changes in their conformation by allosteric effects, could propagate
across networks of multi-complex assemblies [25,29]. Then, allostery is proposed as a fundamental
regulatory device of the cell, used to modulate its activity in response to external and internal stimuli.
Depending on the stimuli that cells receive, multi-scale spatial cellular organization could shape
signaling, and coordinate cellular behavior, through protein clusters [30]. The coordination of the
activities and responses of the cell to its environment could emerge from pre-organized assemblies
across the cell with different length scales. Together, these conditions provide a framework of a spatial
organization of signaling cascades, where signaling could proceed through intermolecular interactions
among and within clusters of proteins [2]. Spatial structure in cells offers a glimpse into the high
Int. J. Mol. Sci. 2015, 16 17197

organization in all levels, from small molecular complexes and assemblies, to local nanoclusters, and
micrometer scales between cells [31]. Likewise, spatial structure of cell signaling systems could be
described in function of dynamic allosteric interactions within and among distinct spatially organized
transient clusters of biomolecules [2].
The existence of intrinsic dynamism as well as static properties of biological systems has been well
established, and although instance the inherent fluctuations within the proteins have been explored in a
specific view, and linked with processes ranging from the acquisition of the three-dimensional
structures to the mechanism of enzymatic action [32,33]. In this focus, protein structure can be
understood as a factor that exerts influence in its surroundings and ultimately in the spatial
organization of the cell. Additional to their functional native structures, proteins or specific regions in
them, acquire other conformations including disordered and partially ordered conformations [34].

2.3. Partially Folded States in Proteins

While the partially folded states in proteins are difficult to conceptualize and their experimental
study is challenging, a wide variety of roles for protein structure disorder has been proposed [35–37].
Disordered proteins are visualized as dynamic assemblies, wherein the atom positions and Ramachandran
angles axis vary significantly over time. Disordered proteins differ from their counterparts structured
in their dynamics. Intrinsically disordered proteins (IDP) represent a challenge to the previously
described structure–function concept, in this case the dominant feature is lack of persistent secondary
and tertiary structure on these proteins [38–40].
The large degree of conformational sampling for IDP gives them significant conformational
entropy, which can be restricted by intra- and inter-molecular interactions. Moreover, it has been
suggested that loss of conformational entropy upon ligand binding originates a weaker binding for IDP,
undergoing disorder-to-order transitions in secondary structure upon ligand interactions [41]. Structural
disorder may span from short segments in specific domains to entire proteins [42], indeed there is
experimental evidence of random conformations in 1539 domains of 694 proteins deposited in a
specialized database (DisProt) [43]. The preponderance of disorder in proteins involved in signaling
networks within higher eukaryotes in comparison with constitutive metabolic proteins or in bacteria [44],
suggests a specific function for IDP, which might be linked to molecular recognition [45,46].
The importance of disorder in proteins is self-evident, as a large portion of molecular interactions
depend on the complementary interaction between structurally organized proteins and IDPs. This
structural condition could confer diverse advantages, such as rapid and specific binding, thus the ability
to carry out some other functions [47]. Interactions among the motifs are usually weak, transient, and
cellular-milieu dependent [48,49]. For instance, the disordered state is present in proteins associated with
transcription, signaling, phosphorylation, RNA processing, ubiquitination, ion transport, cytoskeletal
organization, cell cycle control, and other highly regulated biological mechanisms [50,51]. From an
evolutionary point of view, it appears that intrinsic disorder in proteins might have been the driving
force behind many of the adaptability processes found in proteins [5,52].
The relevance of the absence of rigid structures in proteins in the establishment of communication
between protein networks is evident in the cases of the so-called, guardian of the genome, protein p53
and the proto-oncogene, c-myc, which have long disordered regions [53,54]. The use of IDPs or
Int. J. Mol. Sci. 2015, 16 17198

unstructured domains in proteins can also prove be deleterious as many of the proteins that are
involved in the most common misfolding diseases are intrinsically disordered.
Likewise, specific domains or complete proteins lacking defined tertiary structures are known to
have the fingerprint to undergo disorder-to-order transitions upon binding to specific or multiple
partners [55,56]. This ability allows the concept of protein disorder to be proposed as an important
feature in the capability of proteins to present regions with switch properties [57–59], condition that
could modulate the function, and respond to specific changes in the surrounding microenvironment.
The basic properties of a switch mechanism must be based on the equilibrium between high specificity
and weak affinity, accompanied by a large conformational entropy decrease [60].
Disorder-to-order transitions in proteins playing normal switching roles in the cell might become
distorted and therefore abolish or transform the normal protein-protein language into an aberrant one.
This is the case for α-synuclein, an important protein found in Lewy bodies in the brain of patients
affected with Parkinson disease [61]. In the situation of prion diseases, the PrP protein was
isolated from amyloid plaques, in which a clear conformational change in secondary structure from
α-helix into β-sheet following a templating mechanism, was recognized as the process that could cause
the misfolding phenomenon [62].

2.4. Amyloid Focus

Cells have developed effective strategies to maintain native protein folding, either through an
ordered three-dimensional structure or the use of disordered domains [37]. However, the amyloid
fibrils which represent self-associated species of peptides and proteins [63], considering their
remarkable structures and properties, are of particular interest. Misfolding of normally soluble peptides
and proteins has been associated with about 50 disorders with a multitude of different symptoms,
in which mechanisms of non-native interactions could form aggregates, including the archetypal
amyloid-like fibril [64,65].
Amyloid state has been proposed as a generic condition, being accessible to different polypeptide
chains, and, unlike the native state, its essential three-dimensional architecture is not encoded by the
amino acid sequence [66]. Determined through X-ray fiber diffraction studies, amyloid fibrils show a
common cross-β pattern that is indicative of a structure mainly β-strand, being oriented perpendicularly
to the fibril axis [63,67,68]. The cross-β architecture provides great stability to the fibrils because it
allows the formation of a continuous arrangement of hydrogen bonds [69], complementary steric
interactions. and the formation of a repetitive structural pattern.
Amyloid structures are relevant not only in the context of disease, but also because their occurrence
challenges in many ways our current understanding of the nature, structure, and evolution of the
functional state of proteins [70–73]. From a wide range of experiments on peptides and proteins, we
now know that the formation of amyloid structures is not a rare phenomenon associated with a small
number of diseases, but rather that it reflects a well-defined structural folding of proteins [66,70,74].
Functional amyloids have been described in organisms such as bacteria [75,76], fungi [77–79],
insects [80,81], and mammals [82,83]. Specific cellular function related with the formation of
amyloid-like fibril structures has been described, suggesting that amyloid deposition can be a common
Int. J. Mol. Sci. 2015, 16 17199

property of the polypeptide chains. The difference between functionality and apparition of toxicity may
depend on the regulatory mechanisms that cells have evolved to modulate their formation.
The transition of a protein from its functional native conformation to amyloid fibrils is a complex
phenomenon that results from the interplay between various elementary processes and a rearrangement
in molecular interactions on precursor proteins. As aggregation produces arrangements of different
oligomers, various types of toxic species can be expected considering a mixture of molecular
populations, indeed almost any misfolded specie is likely to have the potential to generate at least some
level of toxicity [84].
A panorama has emerged over the past 15 years that mature amyloid fibrils are not primary toxic
compounds, but molecular species which are precursors to their formation, such as oligomers. Possibly
in the primary mechanism of fibril formation, the exposure of hydrophobic residues is critical for the
nucleation process, a condition that would favor the formation of intermolecular contacts, offering
thermodynamic stability for the amyloid structure (Figure 2) [85]. Although many of the protein
characteristics that preclude amyloid formation are encoded by their amino acid sequences, the
elucidation of this code has enabled the identification of factors that determine the aggregation
propensity of proteins [86,87].

Figure 2. Cell mechanisms that control protein structure. Alterations in protein structure
during folding can result in anomalous interactions with inner membranes through the
exposition of hydrophobic surfaces. Cellular mechanisms, such as proteasome activity and
autophagy, could reduce toxic effects of these molecules, and ultimately prevent cell
damage. Likewise, these processes occur during physiological protein turnover (Adapted
from [6]).
Int. J. Mol. Sci. 2015, 16 17200

Under the exposition of hydrophobic patches to the aqueous medium, proteins might find a stable
conformation to decrease major toxic effects through the formation of amyloid structures. Misfolded
proteins tend to expose hydrophobic regions, which are normally hidden from the aqueous environment,
either by folding characteristics, insertion into membranes or by the support of accessory molecules.
This exposure may cause cytotoxic effects, especially associated to anomalous interaction with lipid
membranes, in turn causing serious damage to the bilayer. Activation of the innate immune response
leading to inflammation has been described as one of the first mechanisms of toxicity associated with
prions, amyloid β (Aβ) peptide, amylin, and other proteins [88].
Likewise, cell damage and death are likely to become widespread only when misfolded
and aggregation-prone species, reach levels that can overwhelm the defensive housekeeping
systems [37,38,89,90]. This breakdown of proteostasis [89,91] can lead to a situation that is known as
protein metastasis, in which initial aggregation events trigger a cascade of pathological processes that
could mark the progression of disease [92].

3. Mechanisms to Conserve Protein Structure

During their functional lifespan, proteins can undergo deviations from their native three-dimensional
structure. In order to maintain the native protein folding, cells have developed various strategies to
accomplish this task, through sophisticated chaperone and quality control networks that can resolve
damage at the level of protein, organelle, or cell (Figure 1). For instance, on the smallest scale, the
integrity of individual proteins is monitored during their synthesis in ribosomes, and coupled with
co-translational chaperone function. On a larger scale, cells use compartmentalized defenses and
networks of communication capable of signaling between cells, and so respond to changes in the
proteome homeostasis [13]. Together, these layered defenses help protect cells from alterations in protein
folding and degradation, avoiding the appearance of misfolded proteins and deleterious events [40].
The organization and evolutionary dynamics of the proteome provide a line of defense in protein
quality control, wherein sequences have evolved to avoid aggregation, most proteins tend to be short
and fold efficiently. This allows the energy-dependent chaperone systems to preferentially protect
long, aggregation-prone, but functionally important proteins. This condition justifies the need for
energetically costly quality control mechanisms to secure protein folding [93,94].

3.1. Chaperone Protein Function

Molecular chaperones are central elements of these quality control systems, as they facilitate protein
biogenesis by assisting polypeptide folding, translocation, and assembly of newly made proteins in
the crowded cellular environment [95,96]. While denatured proteins can refold in vitro without
auxiliary factors, in the crowded molecular cell environment highly specific folding machinery is
required, considering that several billion of protein molecules could exist at concentrations from 50 to
300 mg/mL [97,98].
The recognition of partially unfolded proteins through exposed hydrophobic patches [99,100]
allows the binding of molecular chaperones to a large variety of different protein folding intermediates,
this condition prevents non-specific protein interactions (Figure 2). Likewise, this chaperone function is
associated with proteins during conditions such as the denaturation processes, oligomeric assemblies,
Int. J. Mol. Sci. 2015, 16 17201

proteins that have been translocated to a cell compartment, and during the assistance of proteolytic
degradation [101]. This offers protection of biological molecules against stress-induced unfolding,
along with support of the proteasome quality-control machinery which recognizes unfolded molecules
and subsequently degrades them [102–104]. Thus, molecular chaperones inhibit the formation of
protein aggregates maintaining proteins in a native conformation [105,106].
Molecular chaperones, also known as stress proteins or heat shock proteins (Hsp) are classified into
families on the basis of molecular weight monomers (Hsp40, Hsp60, Hsp70, Hsp90, Hsp110 and small
Hsp), although most of them exist as oligomers. Based on the sort of interaction with client proteins,
chaperones are also classified in holdases, foldases, and disaggregases. Holdases are ATP-independent
proteins, which can recognize and stabilize partially folded proteins, preventing their aggregation and
presenting client proteins to foldases. The ATP-dependent foldases are directly involved in protein
folding. Disaggregases are also ATP-dependent, which disaggregate the client protein aggregates and
transfer the partially folded proteins to a holdase and/or foldase [107,108].
Chaperones Hsp70, Hsp90 and Hsp60 recognize hydrophobic side chains exposed to water
environment in unfolded proteins. This activity is achieved with the support of ATP-independent
chaperones, and the small Hsp (Hsp10 and Hsp40), which function as holdases, and are denominated
co-chaperones. Binding of chaperones to these hydrophobic regions temporarily blocks protein
aggregation, while ATP hydrolysis is important to allow folding of client unfolded proteins [101,109].
Although Hsp70 and Hsp60 share this mechanism to perform their function, they show a significant
difference because Hsp70 releases the client protein for its folding in solution, while Hsp60 form
a cylindrical multimeric complex within which the folding of client protein occurs. The Hsp10
functions as a lid of the Hsp60 complex to close its cavity, whereas the hydrolysis of ATP induces
conformational changes in the inner surface of the cylindrical complex, allowing the folding of client
protein [110]. Hsp60 with the support of its co-chaperone Hsp10 is responsible for folding and the
refolding ATP-dependent, mechanism that allow up to 30% of folding in cells [111].
Chaperones of the Hsp70 family are regulated by co-chaperone Hsp40, preventing the aggregation
of unfolded proteins. This process is involved in folding of proteins that have been translocated to a
cellular compartment, in turn regulate the heat shock response, and disassemble multimeric protein
complexes [111]. Hsp70 and Hsp90 are present in abundance within the ER.
Immunoglobulin binding protein (BiP) also known as GRP78 is the major Hsp70. Specifically, BiP
binds short stretches of hydrophobic residues exposed to the aqueous environment in unfolded proteins,
its activity reduces the effective concentration of aggregation-prone sequences. BiP allows progression
of folding through release and rebinding cycles in unfolded protein substrates, consuming ATP during
the process. With each release cycle, the client protein has the opportunity to fold. In this sense,
folding competes with chaperone binding, and both processes offer the thermodynamic stability to
proteins to acquire native structure [15,112].
Heat shock response (HSR) is coupled with the disposition of denatured proteins in the
cytosol [113,114]. The transcription factor heat shock factor 1 (HSF1) is a key molecule in the
coordination of HSR. This transcription factor is activated during cellular stress induced by the
presence of unfolded proteins, and leads to the transcription of chaperones and other genes that
modulate the folding. Under normal conditions, HSF1 in a monomeric inactive form is bound to the
cytosolic Hsp70 and Hsp90. However, when the load of unfolded proteins increases, these chaperones
Int. J. Mol. Sci. 2015, 16 17202

will dissociate from HSF1 and be recruited to unfolded protein localization. Then, HSF1 trimerizes
and translocates to the nucleus, where it is post-translationally modified by phosphorylation, and
activates the transcription of HSP genes by binding to DNA containing heat shock elements [115].
Although, most knowledge about chaperone function comes from studies of chaperones with
well-defined three-dimensional structures such as Hsp70 and Hsp90 [116], information regarding
client-protein interactions which depend on large ATP-driven conformational rearrangements and
interactions with co-chaperones is lacking [117,118]. Regions lacking defined three-dimensional
structures in chaperones have shown to be critical for the function of certain chaperones, because they
could modulate direct interactions with target proteins [116,119]. The lack of an ordered structure in
regions of chaperones has also been suggested to be important for their ability to bind multiple
aggregation-sensitive client proteins [35]. Disordered chaperones could represent a unique
structure-function relationship, because their structural adaptability enables them to interact with
structurally diverse targets through direct molecular contacts [116]. Disordered regions of proteins
could assume diverse conformations upon binding to different partner proteins [35], and their
chaperone functions could be extended.

3.2. Endoplasmic Reticulum Stress

Protein folding assistance through chaperones is complemented by the role of certain organelles.
Considering that about one-third of the human proteome is synthesized in ER and transits to membrane
compartments, the ER is an organelle that plays key roles in cell homeostasis, such as protein folding
in the protein secretory pathway, lipid biosynthesis, and calcium storage. Lumen of ER is the major
site for protein folding in the cell as it contains a variety of molecular chaperones and protein-folding
enzymes [120]. Therefore, only properly folded proteins are packaged into ER vesicles for further
transport to destination sites [121]. Proteins enter to ER as nascent, unfolded polypeptides at rates that
can vary widely within the cell depending on the requirements of metabolic conditions [122]. When
this machinery is overwhelmed by the increased demand of protein folding capacity, cells suffer a
condition known as ER stress, a consequence of accumulation of unfolded or misfolded proteins in the
lumen [15].
Through a network of intracellular signaling pathways that maintain the folding capacity [122],
UPR is used to re-establish the homeostasis of protein folding function in ER [15]. UPR regulates
translation and gene transcription to reduce the protein-folding load, in turn, increasing the folding
capacity to contend with stress conditions. Distinct downstream signaling pathways are modulated for
UPR signal transducers, three branches operate in parallel, whereas each branch is defined by a
specific transmembrane ER-resident signaling component: IRE1 (inositol requiring enzyme 1), PERK
(PKR-like ER kinase), and ATF6 (activating transcription factor 6). Synthesis of ER resident
chaperones and folding catalysts, is induced to increase the folding capacity; and global mRNA
translation is attenuated to decrease the folding load [123]. Likewise, through a process called
ER-associated degradation (ERAD) misfolded proteins could be retained in the ER and retrotranslocated
into the cytosol for proteasomal degradation [124]. UPR operates within the context of a translocation
machinery that is compartmentalized between cytoplasm and ER [125].
Int. J. Mol. Sci. 2015, 16 17203

ATF6 is a transcription factor that is initially synthesized as an ER-resident transmembrane protein

bearing a large ER-luminal domain. Upon accumulation of unfolded proteins, ATF6 is packaged into
transport vesicles that pinch off the ER and deliver it to the Golgi apparatus [126]. ATF6 is processed
by two proteases, S1P and S2P (site-1 and site-2 protease), that sequentially remove the luminal
domain and the transmembrane anchor, respectively [127,128]. The liberated N-terminal cytosolic
fragment, ATF6 (N), then moves into the nucleus to activate UPR target genes involved in protein
folding, such as BiP, the protein disulfide isomerase, and GRP94 (glucose-regulated protein 94), a
chaperone of the Hsp90 family [14].
The second branch of the UPR is coordinated by PERK. When it is activated upon sensing ER
stress, PERK oligomerizes and phosphorylates itself and the ubiquitous translation initiation factor
eIF2α, then indirectly inactivates eIF2 and inhibits the global translation of mRNA, conditions that
ameliorate the ER stress [14]. Concomitantly, translation of the transcription factor ATF4 is induced,
which promotes transcription of two important target genes, CHOP (transcription factor C/EBP
homologous protein) and GADD34 (growth arrest and DNA damage-inducible 34). CHOP is a
transcription factor that controls genes encoding components involved in apoptosis. GADD34 encodes
a PERK-inducible regulatory subunit of the protein phosphatase PP1C that counteracts the action of
PERK by dephosphorylating eIF2α [14,129,130]. Then, this pathway has an intrinsic feedback regulation.
The best-studied branch of UPR is IRE1, which transmits UPR signaling through a bifunctional
transmembrane kinase/endoribonuclease that splices mRNA through a non-conventional mechanism.
Binding of unfolded proteins triggers conformational changes following lateral oligomerization on the
ER membrane, which in turn activates the IRE1 ribonuclease activity. Furthermore, IRE1 processes
the mRNA of transcription factor XBP1 (X-box binding protein 1) [14] (Figure 3). This active,
processed form of the transcription factor XBP1s controls expression of genes with X-box elements in
their promoters, genes encoding ER chaperones and folding catalysts [131–134]. Additionally, the
IRE1/XBP1 pathway is essential to activate genes which carry out ERAD functions [135], promoting
the development of an elaborate ER response [136]. Active conformation of the kinase domain of
IRE1 has been revealed through its crystal structure, to be an oligomeric assembly [122]. Indeed
ribonuclease activity of IRE1 has been described to be proportional to the extent of IRE1
oligomerization [137].
UPR represents a focal point where different sources of stress converge, and stress signaling is
coordinated within tissue hierarchies and further integrated [14]. Several activated transcription factors
generated by UPR enter the nucleus and activate the production of their target genes, this mechanism
establishes a feedback loop that relieves the ER stress by supplying more ER protein-folding capacity
according to cell requirements [138]. In addition to linear information flow, the three branches of UPR
transmit information to each other through a phenomenon known as cross-talk to fully integrate the
signaling networks [139]. Nodes of interaction and communication between proteins required for
cellular function should be highly regulated.
More experimental evidence is needed to fully understand specific thresholds necessary for the
activation of stress signaling pathways, and in turn when homeostasis is reached again, the mechanisms
that allow these responses to be turned off. For instance, IRE1 and PERK signaling duration is critical to
determine the fate of cells during prolonged stress [140]. Therefore, when homeostasis cannot be
Int. J. Mol. Sci. 2015, 16 17204

re-established, UPR switches from a protective mechanism to a cytotoxic response [141], indeed UPR
can function as an apoptotic executor which decreases cell viability (Figure 3).
Taking into account that ER must manage folding and modification of proteins in concentrations
surpassing 100 mg/mL [142], recognition of unfolded proteins must be a highly precise mechanism
to initiate the correct cellular response. Additionally, other mechanisms constitute a stress adaptation
pathway that could reestablish homeostasis of proteins.

Figure 3. Activation of the inositol requiring enzyme 1 (IRE1) branch of the unfolded
protein response (UPR) pathway is tightly controlled. (A) Schematic representation of
IRE1 oligomerization and cellular response induced by unfolded proteins. The structure
employed was obtained from the protein data bank (PDB) access code: 3fbv;
(B) Uncontrolled protein aggregation in the formation of amyloid fibrils; (C) Effect of the
overactivation of IRE1 branch on cellular homeostasis. Adapted from reference [141];
(D) Structural representation of IRE1 with a focus on the surface of interaction between
monomers. Inset: sterol binding site, with two bound molecules of quercetin. Structure was
obtained from PDB access code: 3LJ0.

3.3. Spatial Compartmentalization of Protein Folding

Connections between loss of proteostasis, protein aggregation phenomenon, and conditions ranging
from ageing to neurodegeneration, underscore the importance of the knowledge of the mechanisms
that cells employ to manage protein misfolding [142]. For instance, it is estimated that up to 15% of
Int. J. Mol. Sci. 2015, 16 17205

nascent chains in human cells are co-translationally tagged for degradation, which emphasizes the
importance of co-translational degradation in protein quality control at the ribosome [143,144]. Cells
must not only promote accurate folding but also must prevent the accumulation of misfolded species
that may arise from inefficient folding, errors in translation, and aberrant mRNAs [145].
An important condition to maintain the functionality of cells is associated with localization of
misfolded or aggregated proteins into specialized compartments that are distinct from the organelles.
Cytotoxicity is avoided through the confinement of misfolded proteins, aggregates, or amyloid like
structures within appropriate and specific subcellular compartments, thus avoiding the subsequently
nucleation of protein aggregates. In this sense, cells have developed mechanisms to solubilize and
fold these proteins, when possible, leaving degradation in defined quality control compartments as a
last-resort mechanism [146].
When quality control machineries fail, such as those previously mentioned, protein-controlled
sequestration into specific compartments represents an alternative cellular defense against proteotoxic
stress [147–149]. Upon proteasome impairment, misfolded proteins are distributed into spatially and
functionally distinct compartments. Evidence has revealed a conserved sequestration of ubiquitinated
proteins into membrane-enclosed juxtanuclear compartments, such as JUNQ (juxtanuclear quality
control compartment). JUNQ is a cellular quality control space wherein soluble misfolded proteins
accumulate for refolding or proteasomal degradation [150].
Under proteotoxic conditions in a cell, several chaperones and proteasome complexes can be
located surrounding the JUNQ compartment, suggesting that JUNQ allows the concentration of
misfolded proteins with chaperones, therefore increasing the probability of refolding, as opposed to
simple uncontrolled degradation [151]. Substrates targeted to JUNQ are primarily soluble proteins,
which are rapidly exchanged with the surrounding environment. When native folding is not reached,
JUNQ substrates are ubiquitinated and recruit proteasome components, triggering protein degradation.
In an important manner, when these mechanisms fail or are diminished, protein misfolding has been
observed by the formation of amyloid fibrils [152,153]. In parallel, diverse strategies have evolved to
maintain the native structure of proteins for prolonged periods of time, and avoiding their conversion
into non-functional misfolded structures [154].
Distinct cytoplasmic structures, spatially distant from JUNQ have been observed to contain large
and highly insoluble aggregates [150]. These compartments denominated IPODs (insoluble protein
deposits) contain insoluble aggregates and amyloid like-fibrils; whereas multiple IPODs can exist at
the same time in cytoplasm, only a single JUNQ is found within each cell. These types of compartments,
underscore their essential function in sequestering proteins and triaging them to re-establish native
protein folding or initiate protein degradation.
While misfolded polypeptides of JUNQ are sequestered in a detergent soluble state, and the
aggregated polypeptides are retained in IPOD [142], other compartments have an important function;
Q-bodies whose formation and processing depend on the cortical ER. The maturation and clearance of
Q-bodies require chaperones Hsp70 and Hsp90 [142].
Biochemical functions of chaperones and their spatial localization within the cell are fundamental to
understand folding impairments during pathological states [142,155], and considering the presence of a
dynamic relationship between damaged and aggregated proteins, the function of these compartments
Int. J. Mol. Sci. 2015, 16 17206

could be very important to maintain the proteostasis in the first instance, and collectively cell
homeostasis [156].

3.4. Proteasome, Structure, and Function

Nascent or newly synthesized polypeptides are predisposed to a high quality control process
associated to their folding, avoiding the accumulation of anomalous proteins. Most studied systems to
maintain proteostasis, are performed by molecular chaperones, as well as two mechanisms of protein
degradation, the ubiquitin-proteasome system and the lysosomal proteolysis through autophagy [157–159].
Proteasome (26S) is a multimeric complex whose function is protein degradation through its
endoprotease activity. Proteasome acts primarily on short-lived proteins with regulatory functions and
on misfolded proteins. Protein degradation is a specific and efficient process, which depends on
ATP. It is involved in functions such as modulation of cell cycle, apoptosis, and cell differentiation,
response to extreme temperature changes, oxidative stress, immune responses, genetic regulation, and
metabolism [160,161].
The 26S proteasome is directly associated to protein degradation via the ubiquitin system. It is
composed of a catalytic subunit comprising a cylindrical central structure with proteolytic activity,
denominated 20S proteasome. Additionally, two regulatory subunits 19S are present, which have
ATPase activity; these are involved in recognition and elimination of ubiquitin chains. Likewise, 19S
subunits participate in the unfolding of client proteins [160] (Figure 4).
The 20S catalytic subunit consists of four complexes arranged in the manner of a ring, forming a
hollow cylindrical structure. Each ring consists of seven different protein subunits, two rings consist of
subunits denominated α (α1–7) located at the ends of the cylindrical structure, the other two rings
consist of subunits called β (β1–7) and are located in the central area [162]. The N-terminal domains of
α-subunits occlude access to the interior of the proteasome, while three of the β subunits have a
protease activity. Therefore, the 20S subunit is a structure α1–7, β1–7, β1–7, α1–7, with proteolytic capacity
and highly conserved between eukaryotes [163,164].
The 19S regulatory subunit is a complex consisting of at least 19 protein subunits, which are
distributed to form the base and lid of the 19S subunit. The base is formed by six proteins with ATPase
activity (Rpt1–Rpt6) that are in contact with α-subunits of the 20S proteasome, and four proteins
lacking ATPase activity (Rpn1, 2, 10, 13). The protein subcomplex which makes up the lid is
integrated by proteins without ATPase activity (Rpn3–9, 11, 12), but contains binding sites for
recognition of ubiquitinated proteins, and maintain a function of deubiquitinase [162,165,166].
Int. J. Mol. Sci. 2015, 16 17207

Figure 4. Scheme of the 26S proteasome. Proteins that make up the base and lid of the 19S
regulatory subunit are shown. The cylindrical portion of the 20S catalytic subunit is shown
in an open conformation, showing the arrangement of α and β proteins identified in orange
and blue, respectively (Adapted from [167]).

The client proteins must have at least four ubiquitins attached to be recognized. Recognition and
anchoring occurs through Rpn10 and Rpn13, which associate with polyubiquitin. Rpn11 accomplishes
the remotion of ubiquitin chains. Proteins with ATPase activity in the 19S subunit perform the
unfolding of ubiquitinated proteins while interacting with the α proteins of the 20S subunit, a condition
that allow its opening, and leads to client proteins into the 20S subunit [163,168,169].
Proteolytic activity of the 20S proteasome subunit lies in the extreme N-terminus of the subunits β1,
β2, and β5 of β-rings. β1 subunit has a caspase activity on amino acids, β2 shows the trypsin activity
on basic amino acids, and β3 subunit a chymotrypsin activity on hydrophobic amino acids. Even more,
in vitro experiments have shown that the 20S subunit alone can present proteolytic activity, generating
peptides of 3–15 residues [167].
Alzheimer’s, Parkinson’s, and Huntington’s diseases are characterized by dysfunction of the
ubiquitin-proteasome system, and accumulation of misfolded proteins in the central nervous system.
Particularly, in Alzheimer’s disease, the patient has two important lesions, extracellular amyloid
plaques, and intraneuronal neurofibrillary tangles formed by Aβ peptide [170,171], which is generated
through sequential cleavage of amyloid precursor protein (APP). In vitro experiments showed that
Aβ40 directly binds to the inside of 20S proteasome and selectively inhibits its chymotrypsin-like
activity [172,173]. More recent evidence shows that Aβ42 also impairs proteasome activity [174,175];
both of them may be endogenous inhibitors of the proteasome. This condition is a consequence of
dysfunction of the ubiquitin-proteasome and its possible association to disease.
Int. J. Mol. Sci. 2015, 16 17208

3.5. Autophagy Mechanism

Autophagy includes a lysosomal degradation pathway, in which cells self-digest their own
components, and has been shown to be essential for survival, differentiation, development, and
homeostasis. Autophagy involves the sequestration of cytoplasmic components in double membrane
autophagosomes, wherein these structures fuse with lysosomes and their cargoes, and are delivered for
degradation and recycling [176].
Mechanisms of autophagy play an important role in removing protein aggregates and organelles that
fail to be degraded by the ubiquitin-proteasome system [177]. Furthermore, the role of autophagy in
maintaining macromolecular synthesis and ATP production is likely a critical mechanism underlying
its evolutionarily conserved pro-survival function [178]. For instance, when cells suddenly undergo a
surge in metabolic demand, autophagy may be needed to generate sufficient intracellular metabolic
substrates to maintain energy levels. This self-digestion process not only provides nutrients to maintain
cellular functions during fasting, but can also relieve cells of superfluous or damaged organelles,
misfolded proteins, and invading microorganisms [179]. Indeed, it has been described that autophagy
could provide an adaptive role to protect organisms against diverse pathologic conditions; which
include: cancer, neurodegeneration, aging, and heart disease [180].
Through this process, cells carry out double-membrane vesicles, denominated autophagosomes, which
could sequester organelles, proteins, or portions of the cytoplasm for delivery to the lysosomes [159].
The core pathway of mammalian autophagy begins with the formation of an isolation membrane
(also called a phagophore), and involves a minimum of five molecular components, including (1) the
AuTophaGy related 1 (Atg1)/unc-51-like kinase (ULK) complex; (2) the Beclin 1/class III
phosphatidylinositol 3-kinase (PI3K) complex; (3) two transmembrane proteins, Atg9, and vacuole
membrane protein 1 (VMP1); (4) two ubiquitin-like protein conjugation systems (Atg12 and
Atg8/LC3); and (5) proteins that drive fusion between autophagosomes and lysosomes. It has been
described that some of these core autophagy pathway components are directly modulated by cellular
stress signals [181,182].
Several functions of autophagy, such as, elimination of defective proteins and organelles, prevention
of protein aggregate accumulation, and clearance of large poly-ubiquitinated proteins, overlap with
those of the ubiquitin-proteasome system; however, pathways leading up to autophagy are uniquely
capable of degrading entire organelles such as mitochondria, peroxisomes, ER, as well as intact
intracellular microorganisms [183]. Unlike proteasomal degradation, the autophagic breakdown of
substrates is not limited by steric conditions, because substrates do not need to be unfolded to pass
through the narrow pore of the proteasomal barrel. Oligomeric and aggregated proteins are poor
substrates for proteasomal degradation, and better targets for autophagic degradation; therefore,
preventing the intracellular accumulation of misfolded proteins and contributing to the proteostasis [183].
Autophagy is also upregulated when cells undergo remodeling events, such as developmental
transitions or to rid themselves of damaging cytoplasmic components, during oxidative stress or
infections [182]. Likewise, autophagy is activated as an adaptive catabolic process in response to
different forms of metabolic stress, including growth factor depletion and hypoxia [183]. Through
autophagy, bulk degradation generates free amino acids and fatty acids that can be recycled or further
processed to maintain ATP production in cells when it is required [182].
Int. J. Mol. Sci. 2015, 16 17209

Nevertheless, alterations in autophagy could result in the accumulation of ubiquitinated and

aggregated proteins, and in turn damaged organelles. In experimental diseases, the self-cannibalistic or,
paradoxically, even the prosurvival functions of autophagy may be deleterious [182]. Autophagosomes
have been observed to accumulate in the brains of patients with diverse neurodegenerative diseases,
including Alzheimer, transmissible spongiform encephalopathies, Parkinson, and Huntington [182].
For instance, autophagosomes accumulate in dystrophic neurons of Alzheimer’s disease patients,
possibly as a result of impairment in autophagolysosomal maturation, consequently contributing to the
accumulation of pathogenic Aβ peptide [184].
On the other hand, the pharmacological activation of autophagy reduces the levels of soluble and
aggregated conformations of mutant huntington, mutated proteins in spinocerebellar ataxia, as well as
mutant forms of α-synuclein and tau. This activation reduces cellular toxicity and their neurotoxicity in
mouse and Drosophila models [185]. Particularly in these models, neuroprotection modulated by
autophagy may be due to a quantitative reduction in the amounts of toxic protein species as well as
anti-apoptotic effects [185].
Autophagy can influence life and death decisions of cells, being cytoprotective or self-destructive;
and being directly linked to apoptotic death pathways. Based on the knowledge of physiological
functions of autophagy, it has been determined that both, basal levels of autophagy and stress-induced
increase of autophagy, are likely determinant in mammalian homeostasis [186].

4. Clinical Focus

Several regulatory and control strategies have evolved in biological systems to protect the
phenomenon of protein folding. Molecular chaperones, protease activities, and molecular factors work
together to refold or remove proteins [187]. When these defensive housekeeping systems of cells are
unable to counteract with these challenges and homeostatic systems are gradually deteriorated,
pathological conditions associated with misfolding become evident [11,90,188]. Even without genetic
defects, protein translation is sufficiently error-prone to allow a missense mutation in proteins
every 1000 to 10,000 amino acids, resulting in defects between 4% and 36% of all new proteins
synthesized [189–191]. This can be tolerated if these proteins can be degraded, but when the load is
excessive, as occurs during cell stress, cell death could appear [192,193].

4.1. Neurodegenerative Diseases

Mechanisms preventing amyloid fibril formation are associated with properties of cell environment,
including the location of proteins within specific compartments [150,194], as well as the presence of
molecular chaperones and degradation mechanisms, such as the ubiquitin–proteasome system and
autophagy [195–199] (Figure 2). Protein misfolding is developed when proteins are unable to attain or
maintain their biologically active conformation [187].
Protein misfolding and formation of toxic aggregates, for instance could affect the proteostasis in
cells to induce ER stress, and in this condition, UPR is required. Likewise, molecular chaperones can
target specific steps in the process that leads to misfolding, specifically inhibiting either primary or
secondary nucleation processes [200], such as the case of the Hsp70 function [201].
Int. J. Mol. Sci. 2015, 16 17210

The disturbance of proteostasis can lead to a situation that is considered a metastasis in proteins,
therefore initial aggregation events trigger a cascade of pathological processes that could mark the
progression of chronic-degenerative diseases [202]. In cases such as Aβ peptide, α-synuclein, and
others peptides, the direct connection between misfolding and the formation of amyloid fibrils, is
determined by structural transitions at the molecular level [40]. Therefore, amyloid formation is
triggered when the protective mechanisms have been exceeded, or due to malfunction of mechanisms
of cell regulation [40].
Protein aggregation and amyloid formation are two fields that have been extensively studied by the
association of amyloid deposition with a range of chronic degenerative disorders, from Alzheimer’s
disease (AD) to diabetes mellitus type 2, many of which are major threats to human health and welfare
in the modern world [203,204]. AD is characterized by cognitive alterations, memory loss, and
behavioral changes. Amyloid plaques and neurofibrillary tangles are the hallmark lesions in the
pathology and both arise from protein misfolding phenomena [205]. In this condition, the Aβ peptide
and tau protein suffer conformational changes that span disordered states and lead to maturation of
toxic aggregates. The presence of such structures leads to ER stress, activating IRE1 and PERK
pathways that active CHOP, conditions reported to induce neuronal death [120]. Likewise, amyloid
precursor protein (APP) and presenilins 1 and 2 have been associated with a familiar form of
Alzheimer. Taking into account their ER membrane location, stress conditions may alter the activity of
presenilin 1 and 2, inducing an increase in the processing of Aβ peptide [206]. In Parkinson disease,
the death of dopaminergic neurons and protein aggregation (Lewy bodies) in different regions of the
brain, is present [207].

4.2. Metabolic Diseases

ER stress is associated with inflammatory and stress signaling pathways, which could exacerbate
metabolic dysfunction, contributing to obesity, insulin resistance, fatty liver, and dyslipidemia [208,209].
The presence of amyloid structures in pancreatic islets of Langerhans is a pathophysiological condition
related with diabetes mellitus type 2. These deposits are composed of a peptide hormone named
amylin [210]. Amylin is normally soluble, and its structure in the monomeric state is natively
disordered. However, secondary structure transitions can be important to attain the three-dimensional
structure found in amyloid fibrils. Aggregation of amylin is associated with an increased response of
ER stress, which leads to dysfunction of pancreatic β cells, apoptosis, and eventually the loss of the
cell mass of islets [211–214]. Likewise, high levels of plasmatic non-esterified fatty acids can
contribute to β cell dysfunction [215]. In our work group, we have been able to establish a potential
relationship of interaction between these important biomolecules [216].
UPR is chronically activated in atherosclerotic related cells, particularly on advanced lesional
macrophages and endothelial cells [217]. Oxidative stress, oxysterols, high levels of intracellular
cholesterol, and saturated fatty acids, are conditions that can lead to prolonged activation of the UPR in
advanced lesions. Likewise, these arterial wall stressors may be associated with obesity, insulin
resistance, and diabetes, all of which promote the clinical progression of atherosclerosis. The potentially
important proatherogenic effect of prolonged ER stress is activation of inflammatory pathways. Even
Int. J. Mol. Sci. 2015, 16 17211

more, prolonged ER stress triggers apoptosis in macrophages, which in turn leads to plaque necrosis if
the apoptotic cells are not rapidly cleared [217].
Conditions associated to atherosclerosis, such as chronic ER stress, affects systemic risk factors at
the level of hepatic lipid metabolism and pancreatic β-cell function [218]. A specific focus has been
performed on signaling modulation through IRE1. Recent developments in understanding how IRE1α
functions to promote cell death versus cell survival at a protein structural level, raise the possibility of
several specific drugs that can block IRE1α-dependent cell death [219,220].

4.3. Cancer and Protein p53

Factors which contribute to a significant increase in protein misfolding incidences are mutations,
thermodynamics, and external stress conditions [187]. The proposal that ER stress signaling could
either be beneficial for tumor growth or play a guardian role to prevent cell transformation is very
important to analyze [221]. Tumor cells are often subjected to major molecular changes due either to
transformation-dependent metabolic demand or to stressful environments, including hypoxia, nutritional
stress or pH stress [222]. For example, one of the conditions of activation of UPR in cancer, has been
attributed to the hypoxic condition in the tumor surrounding environment [223].
Involvement of PERK and IRE1 arms of the UPR in tumor growth has been broadly
characterized [222]. In these conditions, ER stress signaling represents an important constituent of
tumor progression and survival [222]. IRE1α enhances angiogenesis and may alter cell adhesion and
migration through regulated IRE1-dependent decay (RIDD). Likewise, cells deficient in XBP1 or
PERK have a large reduction in their ability to form solid tumors in mice models. In fact, negative
regulation of chaperone activity has been investigated as an anticancer strategy [224,225].
Expression of components of the ER protein-folding machinery, such as BiP, has also been suggested
to promote tumor progression, cell survival, metastasis, and resistance to chemotherapy [226]. Strategies
to downregulate BiP in models of cancer or through the use of inhibitors of the ATP-binding domain
have great cytotoxic potential [227–232].
On the other hand, p53 is a transcription factor with an essential role in guarding cells responses to
various stress signals, through the induction of cell cycle arrest, and apoptosis as well as effects that
are independent of its ability to transactivate gene expression [233]. Mutation of the tumor suppressor
p53 is the most frequent genetic alteration in human cancer [234]. The majority of the mutations occur
in the DNA-binding domain of the p53 (residues 102–292), which result in loss of DNA binding.
Zinc binding, coordinated by H179, C176, C238, and C242, is critical for maintain the native folding of
p53 and requires reduction of thiol groups on cysteines. Residues from the loop-sheet-helix motif
interact in the major groove of the DNA, while an arginine from one of the two large loops interacts in
the minor groove. Loops and the loop-sheet-helix motif represent the conserved regions of the core
domain, and contain the majority of the p53 mutations identified in tumors [235]. In this sense, several
mutations induce conformational changes in the DNA binding surface [236] although destabilized
mutants of p53 can be stabilized by the binding of other molecules [237,238].
Consider that cellular and extracellular spaces are highly saturated environments that allow a wide
variety of interactions between molecules. This feature, often referred to as macromolecular saturated
environment could have important consequences in the thermodynamics of molecules, affecting the
Int. J. Mol. Sci. 2015, 16 17212

conformational states of proteins [239], and then proteostasis. Even more, according to scientists
working in different fields of knowledge, nature appears to have employed disorder to create high
levels of organization. In some cases nature seems to have created disorder, when there is, in the first
place a lack of it [240]. This situation extrapolated to the role of proteins and their association with
disease, could find their origin in the way proteins carry out many structural changes, employing finely
tuned disorder-to-order transitions [34].

5. Protein Folding in Drug Development

In all organisms, energy and nutrient management requires the highly regulated and coordinated
operation of many homeostatic systems. Much of the development and evolution of these systems
has taken place in a different environment to the one we now experience as modern humans,
which includes excess nutrients, new dietary components, lack of physical activity, and an increased
life span [209]. In fact, the requirements for the timespan as well as the magnitude of adaptive
responses have dramatically increased due to rises in life expectancy and a chronic lifetime exposure to
the stress signals [209].
Opportunities for the development of effective therapies against protein-aggregation disorders lie in
the discovery of molecules that decrease the concentrations and formation rates of anomalous protein
assemblies or that enable our natural defenses to maintain their efficacy for longer periods of time [40].
A key milestone in the development of any new therapy is the selection of appropriate molecular targets.

5.1. Strategies Focus on Amyloidosis

Pharmacological strategies for effective therapy in the treatment of diseases associated with protein
folding, might consider the following conditions: inducing the stabilization of native state, reducing the
concentration of aggregation-prone species, blocking the nucleation and growth of aggregates; in
general conditions that are able to reduce the risk of aggregation. Another strategy is optimization of
cell defense mechanisms to maintain their efficacy for longer periods of time, using molecules that can
act as pharmacological chaperones. As already suggested, the most effective procedures for the
prevention and treatment of misfolding diseases, are likely to be those that address the earliest events
in their development [92,241].
Stability of proteins and design of sequences that can efficiently acquire a globular structure,
have been considered to be one of the factors that prevent the conversion of a globular protein into
amyloid-like fibrils. It has been demonstrated that loss of stability in the native state is a primary
mechanism by which mutations promote their pathogenic effects in some hereditary forms of
amyloidosis [242]. For example, nature has used strategies to reduce the number of patterns that favor
the formation of β-structure, alternating groups of hydrophobic and hydrophilic residues, as well as
maintain a high net charge in the sequence, place strategically charged amino acids through the
sequence, generate short β-strands on the edges of large β-sheets, incorporate proline residues, and
cover β-sheets with α-helices [81,83,243,244].
A representative example in this topic is the drug tafamidis (Fx-1006A), a small molecule that acts
as a pharmacological chaperone of transthyretin (TTR), stabilizing the native TTR and some variants.
TTR tetramer dissociation has been described as the rate-limiting step in the amyloid formation
Int. J. Mol. Sci. 2015, 16 17213

cascade. Tetramer dissociation is followed by dimer dissociation yielding unstable monomers. TTR
monomers easily unfold leading to spontaneous self-assembly into amyloid fibrils [245]. Tafamidis is the
first disease modifying pharmacological treatment available to treat familial amyloid polyneuropathy.
In fact, fibril formation has been demonstrated as a mechanism for sequestration of oligomeric species,
in a way that cells reduce toxicity [246].

5.2. Chemical Chaperones

Identification of molecule regulators of the UPR signaling as potential therapeutic strategies to treat
protein misfolding and other human diseases, results in a promising approach. UPR is considered
a target for drug discovery because of emerging evidence from animal models indicating its contribution
to diverse diseases, including cancer, metabolic diseases, diabetes, neurodegenerative disorders,
inflammation, liver dysfunction, and brain and heart ischaemia [247].
Development of drugs that interfere with ER stress, have wide therapeutic potential. Five groups of
strategies according to their mechanism of action have been characterized: compounds directly binding
to ER stress molecules, chemical chaperones, inhibitors of protein degradation, antioxidants, and drugs
affecting calcium signaling. Treatments are generally inhibitory, also lead to increased viability, except
when applied to cancer cells [248].
Chemical chaperones are described as low-molecular mass compounds that stabilize the folding of
proteins and buffer abnormal protein aggregation. In this case, chemical chaperones have been shown
to improve ER function, through diminishing protein misfolding events. The most studied chemical
chaperones in a disease context are 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA),
which have been approved by regulatory authorities for primary biliary cirrhosis (4-BPA) and urea
cycle disorders (TUDCA) [249].
In animal models of obesity, chemical chaperones reduced ER stress in the liver of mouse,
improved insulin sensitivity and glucose homeostasis [250], and reversed leptin resistance [251].
Treatment with 4-PBA also improved glucose tolerance in patients with insulin-resistance [252], and
TUDCA partially restored insulin sensitivity in liver and muscle [253].
High-throughput screening for IRE1 modulators has identified plant-derived flavonols as activators
of IRE1 sensors, as well as possible new regulatory sites of interaction. Docking of small-molecule
libraries suggests the presence of a pocket associated with dimerization/oligomerization of IRE1
including a binding site to sterols, which could represent an important binding site for the regulation of
IRE1 signaling [254,255]. Comparative and systematic studies are needed in a better way to define
the real therapeutic value of manipulating ER stress levels, in addition to outlining possible side
effects [209].
Likewise, approaches through compounds that bind and stabilize mutants of p53 have been
performed. Upon screening of a library of over 100,000 compounds and further optimizing of the hits,
compound 7 (CP-31398) was shown to promote the conformational stability of wild-type p53
DNA-binding domain and that of full-length p53 [237,256]. Screening of the diversity set from the
National Cancer Institute led to the discovery of some chemical chaperones with mutant
p53-reactivating capacity: compound 854 known as PRIMA-1 (p53 reactivation and induction of
massive apoptosis) and compound 954 known as MIRA-3 (mutant p53 dependent induction of
Int. J. Mol. Sci. 2015, 16 17214

rapid apoptosis). Additional screening of small compound libraries also identified compound 1055
denominated STIMA-1 (SH group-targeting compound that induces massive apoptosis) [187].

5.3. Final Considerations

Three-dimensional structure of proteins in general, and specifically proteins that participate in UPR
could provide critical information for the development of new pharmacological treatments, this
approach may be incomplete, since the disordered domains of proteins involved in UPR and
chaperones, have been proven to be critical for their function. Likewise, complex pathways to ensure
proteostasis in different subcellular compartments, defined as unfolded protein responses have evolved
in the cytosol and mitochondria, which are finely coordinated and require close communication with
the nucleus [248]. Even more, the UPR has demonstrated impact on various immune cells, in which
it regulates the secretion of pro-inflammatory cytokines and innate immunity signals [254]. These
conditions reflect the complexity of cell physiology that might be considered for drug development.
Whereas cellular and extracellular spaces are highly saturated environments [97,98] which allow a
wide variety of interactions between molecules, not all biologically active compounds have the desired
physicochemical properties to be a drug, which must be sufficiently lipophilic to be absorbed, maintain
polar properties to cross the gastrointestinal wall, and have a vulnerable chemical functionality,
then molecules can be targeted by liver catabolic systems [26]. Without doubt a more complex
understanding of the threshold of responses that occur within cells to sustain the proteostasis, implies a
greater understanding of the regulatory mechanisms that regulate protein folding, which will result in
an increase in tools that could be the basis to modulate the functional activity of therapeutically
important proteins.

6. Conclusions

Mechanisms that modulate protein folding within the highly saturated cellular environment, which
are regulated by a highly sophisticated network of communication between proteins, reflect the
complexity of cellular processes. These features span several molecular hierarchies, from the use of
small disordered regions within intricate three-dimensional structures, and an effective folding
phenomenon to keep hidden the hydrophobic domains. Likewise required are the activity of highly
organized molecules such as chaperones, and the participation of pathways associated with complete
degradation of organelles, to maintain the homeostasis of proteins as a whole. Evidence indicates that
nearly all large, subcellular processes, from the organelles to ribosomes, may have specific ways of
sensing the proteome and reacting to proteotoxic stress, and so that every step in the life of proteins is
under close scrutiny. Therefore, the insights into the features of the functional conformations of
proteins, the environments in which they work, and the ways that cellular defense mechanisms
normally function so effectively together to maintain protein homeostasis, can expand the possibilities
for better treatments against human diseases.
Int. J. Mol. Sci. 2015, 16 17215

Acknowledgments

The authors recognized the help of Julia Dolores Estrada Guzmán, and we want to thank
Nadia Gutiérrez Quintanar and Pedro Alberto Méndez Jaime for editorial support. This work was supported
by Coordinación de Posgrado e Investigación, Universidad Autónoma de Baja California, and by Programa
para el Desarrollo Profesional Docente para el tipo Superior, Secretaria de Educación Pública.

Author Contributions

Victor García-González conceived the idea and wrote the manuscript; José Fernando Díaz-Villanueva
was involved in discussion and helped of manuscript preparation; and Raúl Díaz-Molina critically
reviewed the manuscript. All authors approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Badyaev, A.V. Stress-induced variation in evolution: From behavioural plasticity to genetic

assimilation. Proc. Biol. Sci. 2005, 272, 877–886.
2. Nussinov, R. The spatial structure of cell signaling systems. Phys. Biol. 2013, 10, 045004.
3. Nussinov, R.; Wolynes, P.G. A second molecular biology revolution? The energy landscapes of
biomolecular function. Phys. Chem. Chem. Phys. 2014, 16, 6321–6322.
4. Rutkowski, D.T.; Hegde, R.S. Regulation of basal cellular physiology by the homeostatic
unfolded protein response. J. Cell Biol. 2010, 189, 783–794.
5. Dobson, C.M. Protein misfolding, evolution and disease. Trends Biochem. Sci. 1999, 24, 329–332.
6. Seong, S.Y.; Matzinger, P. Hydrophobicity: An ancient damage-associated molecular pattern that
initiates innate immune responses. Nat. Rev. Immunol. 2004, 4, 469–478.
7. Campioni, S.; Mannini, B.; Zampagni, M.; Pensalfini, A.; Parrini, C.; Evangelisti, E.; Relini, A.;
Stefani, M.; Dobson, C.M.; Cecchi, C.; et al. A causative link between the structure of aberrant
protein oligomers and their toxicity. Nat. Chem. Biol. 2010, 6, 140–147.
8. Cheon, M.; Chang, I.; Mohanty, S.; Luheshi, L.M.; Dobson, C.M.; Vendruscolo, M.; Favrin, G.
Structural reorganisation and potential toxicity of oligomeric species formed during the assembly
of amyloid fibrils. PLoS Comput. Biol. 2007, 3, 1727–1738.
9. Olzscha, H.; Schermann, S.M.; Woerner, A.C.; Pinkert, S.; Hecht, M.H.; Tartaglia, G.G.;
Vendruscolo, M.; Hayer-Hartl, M.; Hartl, F.U.; Vabulas, R.M. Amyloid-like aggregates sequester
numerous metastable proteins with essential cellular functions. Cell 2011, 144, 67–78.
10. Narayan, P.; Ganzinger, K.A.; McColl, J.; Weimann, L.; Meehan, S.; Qamar, S.; Carver, J.A.;
Wilson, M.R.; St George-Hyslop, P.; Dobson, C.M.; et al. Single molecule characterization of
the interactions between amyloid-β peptides and the membranes of hippocampal cells. J. Am.
Chem. Soc. 2013, 135, 1491–1498.
11. Liu, Y.; Ye, Y. Proteostasis regulation at the endoplasmic reticulum: A new perturbation site for
targeted cancer therapy. Cell Res. 2011, 21, 867–883.
Int. J. Mol. Sci. 2015, 16 17216

12. Princiotta, M.F.; Finzi, D.; Qian, S.B.; Gibbs, J.; Schuchmann, S.; Buttgereit, F.; Bennink, J.R.;
Yewdell, J.W. Quantitating protein synthesis, degradation, and endogenous antigen processing.
Immunity 2003, 18, 343–354.
13. Zhong, F.; Yang, D.; Hao, Y.; Lin, C.; Jiang, Y.; Ying, W.; Wu, S.; Zhu, Y.; Liu, S.; Yang, P.;
et al. Regular patterns for proteome-wide distribution of protein abundance across species.
PLoS ONE 2012, 7, e32423.
14. Walter, P.; Ron, D. The unfolded protein response: From stress pathway to homeostatic
regulation. Science 2011, 334, 1081–1086.
15. Chambers, J.E.; Marciniak, S.J. Cellular mechanisms of endoplasmic reticulum stress signaling
in health and disease. 2. Protein misfolding and ER stress. Am. J. Physiol. Cell Physiol. 2014,
307, C657–C670.
16. Manié, S.N.; Lebeau, J.; Chevet, E. Cellular mechanisms of endoplasmic reticulum stress
signaling in health and disease. 3. Orchestrating the unfolded protein response in oncogenesis:
An update. Am. J. Physiol. Cell Physiol. 2014, 307, C901–C907.
17. Sano, R.; Reed, J.C. ER stress-induced cell death mechanisms. Biochim. Biophys. Acta 2013,
1833, 3460–3470.
18. Hinault, M.P.; Ben-Zvi, A.; Goloubinoff, P. Chaperones and proteases: Cellular fold-controlling
factors of proteins in neurodegenerative diseases and aging. J. Mol. Neurosci. 2006, 30, 249–265.
19. Huang, K. Lectures on Statistical Physics and Protein Folding, 1st ed.; World Scientific:
Hackensack, NJ, USA, 2005; pp. 1–100.
20. Dobson, M.C. Protein chemistry: In the footsteps of alchemists. Science 2004, 304, 1259–1262.
21. Anfinsen, C.B. Principles that govern folding of protein chains. Science 1973, 181, 223–230.
22. Dyson, H.J.; Wright, P.E. Intrinsically unstructured proteins and their functions. Nat. Rev. Mol.
Cell Biol. 2005, 6, 197–208.
23. Uversky, V.N.; Dunker, A.K. Understanding protein non-folding. Biochim. Biophys. Acta 2010,
1804, 1231–1264.
24. Vuillon, L.; Lesieur, C. From local to global changes in proteins: A network view. Curr. Opin.
Struct. Biol. 2015, 31, 1–8.
25. Nussinov, R.; Tsai, C.J. Allostery in disease and in drug discovery. Cell 2013, 153, 293–305.
26. Lipinski, C.; Hopkins, A. Navigating chemical space for biology and medicine. Nature 2004,
432, 855–861.
27. Zuiderweg, E.R.; Bertelsen, E.B.; Rousaki, A.; Mayer, M.P.; Gestwicki, J.E.; Ahmad, A.
Allostery in the Hsp70 chaperone proteins. Top. Curr. Chem. 2013, 328, 99–153.
28. Tompa, P. Multisteric regulation by structural disorder in modular signaling proteins: An
extension of the concept of allostery. Chem. Rev. 2014, 114, 6715–6732.
29. Deshpande, S.; Pfohl, T. Real-time dynamics of emerging actin networks in cell-mimicking
compartments. PLoS ONE 2015, 10, e011652.
30. Manz, B.N.; Groves, J.T. Spatial organization and signal transduction at intercellular junctions.
Nat. Rev. Mol. Cell Biol. 2010, 11, 342–352.
31. He, X.; Zhang, J. Why do hubs tend to be essential in protein networks? PLoS Genet. 2006, 2, e88.
Int. J. Mol. Sci. 2015, 16 17217

32. Verdian Doghaei, A.; Housaindokht, M.R.; Bozorgmehr, M.R. Molecular crowding effects on
conformation and stability of G-quadruplex DNA structure: Insights from molecular dynamics
simulation. J. Theor. Biol. 2015, 364, 103–112.
33. Karplus, M.; McCammon, A.J. Molecular dynamics simulations of biomolecules. Nat. Struct. Biol.
2002, 9, 646–652.
34. Mendoza-Espinosa, P.; García-González, V.; Moreno, A.; Castillo, R.; Mas-Oliva, J.
Disorder-to-order conformational transitions in protein structure and its relationship to disease.
Mol. Cell. Biochem. 2009, 330, 105–120.
35. Bardwell, J.C.; Jakob, U. Conditional disorder in chaperone action. Trends Biochem. Sci. 2012,
37, 517–525.
36. Górecki, A.; Bonarek, P.; Górka, A.K.; Figiel, M.; Wilamowski, M.; Dziedzicka-Wasylewska, M.
Intrinsic disorder of human Yin Yang 1 protein. Proteins 2015, doi:10.1002/prot.24822.
37. García-González, V.; Mas-Oliva, J. Amyloidogenic properties of a D/N mutated 12 amino
acid fragment of the C-terminal domain of the Cholesteryl-Ester Transfer Protein (CETP). Int. J.
Mol. Sci. 2011, 12, 2019–2035.
38. Guharoy, M.; Pauwels, K.; Tompa, P. SnapShot: Intrinsic structural disorder. Cell 2015, 161,
1230–1230.
39. Forman-Kay, J.D.; Mittag, T. From sequence and forces to structure, function, and evolution of
intrinsically disordered proteins. Structure 2013, 21, 1492–1499.
40. García-González, V.G.; Mas-Oliva, J. El Concepto de Enfermedad Asociado a la Conformación
de Proteínas, 1st ed.; Universidad Nacional Autónoma de México and El Manual Moderno:
Mexico City, Mexico, 2012; pp. 11–21.
41. Dunker, A.K.; Silman, I.; Uversky, V.N.; Sussman, J.L. Function and structure of inherently
disordered proteins. Curr. Opin. Struct. Biol. 2008, 18, 756–764.
42. Tompa, P.; Fuxreiter, M.; Oldfield, C.J.; Simon, I.; Dunker, A.K.; Uversky, V.N. Close
encounters of the third kind: Disordered domains and the interactions of proteins. BioEssays
2009, 31, 328–335.
43. Sickmeier, M.; Hamilton, J.A.; LeGall, T.; Vacic, V.; Cortese, M.S.; Tantos, A.; Szabo, B.;
Tompa, P.; Chen, J.; Uversky, V.N.; et al. DisProt: The database of disordered proteins.
Nucleic Acids Res. 2007, 35, D786–D793.
44. Tantos, A.; Han, K.H.; Tompa, P. Intrinsic disorder in cell signaling and gene transcription.
Mol. Cell. Endocrinol. 2012, 348, 457–465.
45. Hilser, V.J.; Thompson, E.B. Intrinsic disorder as a mechanism to optimize allosteric coupling in
proteins. Proc. Natl. Acad. Sci. USA 2007, 104, 8311–8315.
46. Ferreon, A.C.; Ferreon, J.C.; Wright, P.E.; Deniz, A.A. Modulation of allostery by protein
intrinsic disorder. Nature 2013, 498, 390–394.
47. Tompa, P.; Szász, C.; Buday, L. Structural disorder throws new light on moonlighting.
Trends Biochem. Sci. 2005, 30, 484–489.
48. Gould, C.M.; Diella, F.; Via, A.; Puntervoll, P.; Gemünd, C.; Chabanis-Davidson, S.;
Michael, S.; Sayadi, A.; Bryne, J.C.; Chica, C.; et al. ELM: The status of the 2010 eukaryotic
linear motif resource. Nucleic Acids Res. 2010, 38, D167–D180.
Int. J. Mol. Sci. 2015, 16 17218

49. Stein, A.; Aloy, P. Contextual specificity in peptide-mediated protein interactions. PLoS ONE
2008, 3, e2524.
50. Ward, J.J.; Sodhi, J.S.; McGuffin, L.J.; Buxton, B.F.; Jones, D.T. Prediction and functional
analysis of native disorder in proteins from the three kingdoms of life. J. Mol. Biol. 2004, 337,
635–645.
51. Lobley, A.; Swindells, M.B.; Orengo, C.A.; Jones, D.T. Inferring function using patterns of
native disorder in proteins. PLoS Comput. Biol. 2007, 3, e162.
52. Dunker, A.K.; Garner, E.; Guilliot, S.; Romero, P.; Albrecht, K.; Hart, J.; Obradovic, Z.;
Kissinger, C.; Villafranca, J.E. Protein disorder and the evolution of molecular recognition:
Theory, predictions and observations. Pac. Symp. Biocomput. 1998, 473–484.
53. Oldfield, C.J.; Meng, J.; Yang, J.Y.; Yang, M.Q.; Uversky, V.N.; Dunker, A.K. Flexible nets:
Disorder and induced fit in the associations of p53 and 14-3-3 with their partners. BMC Genomics
2008, 9, S1.
54. Andresen, C.; Helander, S.; Lemak, A.; Farès, C.; Csizmok, V.; Carlsson, J.; Penn, L.Z.;
Forman-Kay, J.D.; Arrowsmith, C.H.; Lundström, P.; et al. Transient structure and dynamics in
the disordered c-Myc transactivation domain affect Bin1 binding. Nucleic Acids Res. 2012, 40,
6353–6266.
55. Tompa, P. Intrinsically unstructured proteins. Trends Biochem. Sci. 2002, 27, 527–533.
56. James, C.; Tawfik, D.S. Conformational diversity and protein evolution—A 60-year-old
hypothesis revisited. Trends Biochem. Sci. 2003, 28, 361–368.
57. Bustos, D.M.; Iglesias, A.A. Intrinsic disorder is a key characteristic in partners that bind 14-3-3
proteins. Proteins 2006, 63, 35–42.
58. Kriwacki, R.W.; Hengst, L.; Tennant, L.; Reed, S.I.; Wright, P.E. Structural studies of
p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: Conformational disorder mediates binding
diversity. Proc. Natl. Acad. Sci. USA 1996, 93, 11504–11509.
59. Dalal, S.; Regan, L. Understanding the sequence determinants of conformational switching using
protein design. Protein Sci. 2000, 9, 1651–1659.
60. Cheng, Y.; LeGall, T.; Oldfield, C.J.; Mueller, J.P.; Van, Y.Y.; Romero, P.; Cortese, M.S.;
Uversky, V.N.; Dunker, A.K. Rational drug design via intrinsically disordered protein.
Trends Biotechnol. 2006, 2, 435–442.
61. Chandra, S.; Chen, X.; Rizo, J.; Jahn, R.; Sudhof, T.C. A broken α-helix in folded α-synuclein.
J. Biol. Chem. 2003, 278, 15313–15318.
62. Fink, A.L. Protein aggregation: Folding aggregates, inclusion bodies and amyloid. Fold. Des.
1998, 3, R9–R23.
63. Eisenberg, D.; Jucker, M. The amyloid state of proteins in human diseases. Cell 2012, 148,
1188–1203.
64. Meredith, S.C. Protein denaturation and aggregation: Cellular responses to denatured and
aggregated proteins. Ann. N. Y. Acad. Sci. 2005, 1066, 181–221.
65. Chiti, F.; Dobson, C.M. Protein misfolding, functional amyloid, and human disease.
Annu. Rev. Biochem. 2006, 75, 333–366.
66. Dobson, C.M. Protein folding and misfolding. Nature 2003, 426, 884–890.
Int. J. Mol. Sci. 2015, 16 17219

67. Sawaya, M.R.; Sambashivan, S.; Nelson, R.; Ivanova, M.I.; Sievers, S.A.; Apostol, M.I.;
Thompson, M.J.; Balbirnie, M.; Wiltzius, J.J.; McFarlane, H.T.; et al. Atomic structures of
amyloid cross-β spines reveal varied steric zippers. Nature 2007, 447, 453–457.
68. Fitzpatrick, A.W.P.; Debelouchina, G.T.; Bayro, M.J.; Clare, D.K.; Caporini, M.A.; Bajaj, V.S.;
Jaroniec, C.P.; Wang, L.; Ladizhansky, V.; Müller, S.A. Atomic structure and hierarchical
assembly of a cross-β amyloid fibril. Proc. Natl Acad. Sci. USA 2013, 110, 5468–5473.
69. Knowles, T.P.; Fitzpatrick, A.W.; Meehan, S.; Mott, H.R.; Vendruscolo, M.; Dobson, C.M.;
Welland, M.E. Role of intermolecular forces in defining material properties of protein
nanofibrils. Science 2007, 318, 1900–1903.
70. De Genst, E.; Chirgadze, D.Y.; Klein, F.A.; Butler, D.C.; Matak-Vinković, D.; Trottier, Y.;
Huston, J.S.; Messer, A.; Dobson, C.M. Structure of a single-chain fv bound to the 17 N-terminal
residues of huntingtin provides insights into pathogenic amyloid formation and suppression.
J. Mol. Biol. 2015, 427, 2166–2178.
71. Greenwald, J.; Riek, R. On the possible amyloid origin of protein folds. J. Mol. Biol. 2012, 421,
417–426.
72. Carny, O.; Gazit, E. A model for the role of short self-assembled peptides in the very early stages
of the origin of life. FASEB J. 2005, 19, 1051–1055.
73. Fowler, D.M.; Koulov, A.V.; Balch, W.E.; Kelly, J.W. Functional amyloid—From bacteria to
humans. Trends Biochem. Sci. 2007, 32, 217–224.
74. Fandrich, M.; Dobson, C.M. The behaviour of polyamino acids reveals an inverse side chain
effect in amyloid structure formation. EMBO J. 2002, 21, 5682–5690.
75. Chapman, M.R.; Robinson, L.S.; Pinkner, J.S.; Roth, R.; Heuser, J.; Hammar, M.; Normark, S.;
Hultgren, S.J. Role of Escherichia coli curli operons in directing amyloid fiber formation.
Science 2002, 295, 851–855.
76. Claessen, D.; Rink, R.; de Jong, W.; Siebring, J.; de Vreugd, P.; Boersma, F.G.; Dijkhuizen, L.;
Wosten, H.A. A novel class of secreted hydrophobic proteins is involved in aerial hyphae
formation in Streptomyces coelicolor by forming amyloid-like fibrils. Genes Dev. 2003, 17,
1714–1726.
77. Mackay, J.P.; Matthews, J.M.; Winefield, R.D.; Mackay, L.G.; Haverkamp, R.G.; Templeton, M.D.
The hydrophobin EAS is largely unstructured in solution and functions by forming amyloid-like
structures. Structure 2001, 9, 83–91.
78. Coustou, V.; Deleu, C.; Saupe, S.; Begueret, J. The protein product of the het-s heterokaryon
incompatibility gene of the fungus Podospora anserina behaves as a prion analog. Proc. Natl.
Acad. Sci. USA 1997, 94, 9773–9778.
79. King, C.Y.; Tittmann, P.; Gross, H.; Gebert, R.; Aebi, M.; Wüthrich, K. Prion-inducing domain
2–114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments. Proc. Natl. Acad.
Sci. USA 1997, 94, 6618–6622.
80. Iconomidou, V.A.; Vriend, G.; Hamodrakas, S.J. Amyloids protect the silkmoth oocyte and
embryo. FEBS Lett. 2000, 479, 141–145.
Int. J. Mol. Sci. 2015, 16 17220

81. Iconomidou, V.A.; Chryssikos, G.D.; Gionis, V.; Galanis, A.S.; Cordopatis, P.; Hoenger, A.;
Hamodrakas, S.J. Amyloid fibril formation propensity is inherent into the hexapeptide
tandemly repeating sequence of the central domain of silkmoth chorion proteins of the A-family.
J. Struct. Biol. 2006, 156, 480–488.
82. Berson, J.F.; Theos, A.C.; Harper, D.C.; Tenza, D.; Raposo, G.; Marks, M.S. Proprotein convertase
cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome
biogenesis. J. Cell Biol. 2003, 161, 521–533.
83. Kobayashi, T.; Urabe, K.; Orlow, S.J.; Higashi, K.; Imokawa, G.; Kwon, B.S.; Potterf, B.;
Hearing, V.J. The Pmel 17/silver locus protein. Characterization and investigation of its
melanogenic function. J. Biol. Chem. 1994, 269, 29198–29205.
84. Stefani, M.; Dobson, C.M. Protein aggregation and aggregate toxicity: New insights into protein
folding, misfolding diseases and biological evolution. J. Mol. Med. 2003, 81, 678–699.
85. García-González, V.; Mas-Oliva, J. Amyloid fibril formation of peptides derived from the
C-terminus of CETP modulated by lipids. Biochem. Biophys. Res. Commun. 2013, 434, 54–59.
86. Fernandez-Escamilla, A.M.; Rousseau, F.; Schymkowitz, J.; Serrano, L. Prediction of
sequence-dependent and mutational effects on the aggregation of peptides and proteins.
Nat. Biotechnol. 2004, 22, 1302–1306.
87. Pawar, A.P.; Dubay, K.F.; Zurdo, J.; Chiti, F.; Vendruscolo, M.; Dobson, C.M. Prediction
of “aggregation-prone” and “aggregation-susceptible” regions in proteins associated with
neurodegenerative diseases. J. Mol. Biol. 2005, 350, 379–392.
88. Matzinger, P. Friendly and dangerous signals: Is the tissue in control? Nat. Immunol. 2007, 8,
11–13.
89. Balch, W.E.; Morimoto, R.I.; Dillin, A.; Kelly, J.W. Adapting proteostasis for disease intervention.
Science 2008, 319, 916–919.
90. Kim, Y.E.; Hipp, M.S.; Bracher, A.; Hayer-Hartl, M.; Hartl, F.U. Molecular chaperone functions
in protein folding and proteostasis. Annu. Rev. Biochem. 2013, 82, 323–355.
91. Vendruscolo, M.; Knowles, T.P.J.; Dobson, C.M. Protein solubility and protein homeostasis:
A generic view of protein misfolding disorders. Cold Spring Harb. Perspect. Biol. 2011, 3, a010454.
92. Knowles, T.P.; Vendruscolo, M.; Dobson, C.M. The amyloid state and its association with
protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 2014, 15, 384–396.
93. Calloni, G.; Chen, T.; Schermann, S.M.; Chang, H.-C.; Genevaux, P.; Agostini, F.; Tartaglia, G.G.;
Hayer-Hartl, M.; Hartl, F.U. DnaK functions as a central hub in the E. coli chaperone network.
Cell Rep. 2012, 1, 251–264.
94. Willmund, F.; del Alamo, M.; Pechmann, S.; Chen, T.; Albanese, V.; Dammer, E.B.; Peng, J.;
Frydman, J. The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein
homeostasis. Cell 2013, 152, 196–209.
95. Gershenson, A.; Gierasch, L.M. Protein folding in the cell: Challenges and progress. Curr. Opin.
Struct. Biol. 2011, 21, 32–41.
96. Hartl, F.U.; Bracher, A.; Hayer-Hartl, M. Molecular chaperones in protein folding and
proteostasis. Nature 2011, 475, 324–332.
97. Asherie, N. Protein crystallization and phase diagrams. Methods 2004, 34, 266–272.
Int. J. Mol. Sci. 2015, 16 17221

98. Finka, A.; Goloubinoff, P. Proteomic data from human cell cultures refine mechanisms of
chaperone-mediated protein homeostasis. Cell Stress Chaperones 2013, 18, 591–605.
99. Reichmann, D.; Xu, Y.; Cremers, C.M.; Ilbert, M.; Mittelman, R.; Fitzgerald, M.C.; Jakob, U.
Order out of disorder: Working cycle of an intrinsically unfolded chaperone. Cell 2012, 148,
947–957.
100. Rosenbaum, J.C.; Fredrickson, E.K.; Oeser, M.L.; Garrett-Engele, C.M.; Locke, M.N.;
Richardson, L.A.; Nelson, Z.W.; Hetrick, E.D.; Milac, T.I.; Gottschling, D.E.; et al. Disorder
targets misorder in nuclear quality control degradation: A disordered ubiquitin ligase directly
recognizes its misfolded substrates. Mol. Cell 2011, 41, 93–106.
101. Mayer, M.P. Gymnastics of molecular chaperones. Mol. Cell 2010, 39, 321–331.
102. Ellis, R.J. Molecular chaperones: Assisting assembly in addition to folding. Trends Biochem. Sci.
2006, 31, 395–401.
103. Saibil, H.R. Chaperone machines in action. Curr. Opin. Struct. Biol. 2008, 18, 35–42.
104. Wandinger, S.K.; Richter, K.; Buchner, J. The Hsp90 chaperone machinery. J. Biol. Chem. 2008,
283, 18473–18477.
105. Tokuriki, N.; Tawfik, D.S. Chaperonin overexpression promotes genetic variation and enzyme
evolution. Nature 2009, 459, 668–671.
106. Rutherford, S.L.; Lindquist, S. Hsp90 as a capacitor for morphological evolution. Nature 1998,
396, 336–342.
107. Slepenkov, S.V.; Witt, S.N. The unfolding story of the Escherichia coli Hsp70 DnaK: Is DnaK a
holdase or an unfoldase? Mol. Microbiol. 2002, 45, 1197–1206.
108. Bosl, B.; Grimminger, V.; Walter, S. The molecular chaperone Hsp104—A molecular machine
for protein disaggregation. J. Struct. Biol. 2006, 156, 139–148.
109. Langer, T.; Lu, C.; Echols, H.; Flanagan, J.; Hayer, M.K.; Hartl, F.U. Successive action of
DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature 1992,
356, 683–689.
110. Horwich, A.L.; Fenton, W.A. Chaperonin-mediated protein folding: Using a central cavity to
kinetically assist polypeptide chain folding. Q. Rev. Biophys. 2009, 42, 83–116.
111. Taldone, T.; Patel, H.J.; Bolaender, A.; Patel, M.R.; Chiosis, G. Protein chaperones: A
composition of matter review (2008–2013). Expert Opin. Ther. Pat. 2014, 24, 501–518.
112. Yan, M.; Li, J.; Sha, B. Structural analysis of the Sil1-Bip complex reveals the mechanism for
Sil1 to function as a nucleotide-exchange factor. Biochem. J. 2011, 438, 447–455.
113. Morimoto, R.I. The heat shock response: Systems biology of proteotoxic stress in aging and
disease. Cold Spring Harb. Symp. Quant. Biol. 2011, 76, 91–99.
114. Vabulas, R.M.; Raychaudhuri, S.; Hayer-Hartl, M.; Hartl, F.U. Protein folding in the cytoplasm
and the heat shock response. Cold Spring Harb. Perspect. Biol. 2010, 2, a004390.
115. Westerheide, S.D.; Anckar, J.; Stevens, S.M., Jr.; Sistonen, L.; Morimoto, R.I. Stress-inducible
regulation of heat shock factor 1 by the deacetylase SIRT1. Science 2009, 323, 1063–1066.
116. Kovacs, D.; Tompa, P. Diverse functional manifestations of intrinsic structural disorder in
molecular chaperones. Biochem. Soc. Trans. 2012, 40, 963–968.
117. Snapp, E.L. Unfolded protein responses with or without unfolded proteins? Cells 2012, 1,
926–950.
Int. J. Mol. Sci. 2015, 16 17222

118. Carrara, M.; Prischi, F.; Nowak, P.R.; Kopp, M.C.; Ali, M.M. Noncanonical binding of BiP
ATPase domain to Ire1 and Perk is dissociated by unfolded protein CH1 to initiate ER stress
signaling. Elife 2015, 4, doi:10.7554/eLife.03522.
119. Foit, L.; George, J.S.; Zhang, B.W.; Brooks, C.L., 3rd; Bardwell, J.C. Chaperone activation by
unfolding. Proc. Natl. Acad. Sci. USA 2013, 110, E1254–E1262.
120. Ozcan, L.; Tabas, I. Role of endoplasmic reticulum stress in metabolic disease and other
disorders. Annu. Rev. Med. 2012, 63, 317–328.
121. Ellgaard, L.; Helenius, A. Quality control in the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol.
2003, 4, 181–191.
122. Korennykh, A.; Walter, P. Structural basis of the unfolded protein response. Annu. Rev. Cell
Dev. Biol. 2012, 28, 251–277.
123. Zhou, J.; Liu, C.Y.; Back, S.H.; Clark, R.L.; Peisach, D.; Xu, Z.; Kaufman, R.J. The crystal
structure of human IRE1 luminal domain reveals a conserved dimerization interface required for
activation of the unfolded protein response. Proc. Natl. Acad. Sci. USA 2006, 103, 14343–14348.
124. Smith, M.H.; Ploegh, H.L.; Weissman, J.S. Road to ruin: Targeting proteins for degradation in
the endoplasmic reticulum. Science 2011, 334, 1086–1090.
125. Voeltz, G.K.; Rolls, M.M.; Rapoport, T.A. Structural organization of the endoplasmic reticulum.
EMBO Rep. 2002, 3, 944–950.
126. Schindler, A.J.; Schekman, R. In vitro reconstitution of ER-stress induced ATF6 transport in
COPII vesicles. Proc. Natl. Acad. Sci. USA 2009, 106, 17775–17780.
127. Haze, K.; Yoshida, H.; Yanagi, H.; Yura, T.; Mori, K. Mammalian transcription factor ATF6 is
synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic
reticulum stress. Mol. Biol. Cell 1999, 10, 3787–3799.
128. Ye, J.; Rawson, R.B.; Komuro, R.; Chen, X.; Davé, U.P.; Prywes, R.; Brown, M.S.; Goldstein, J.L.
ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs.
Mol. Cell 2000, 6, 1355–1364.
129. Marciniak, S.J.; Yun, C.Y.; Oyadomari, S.; Novoa, I.; Zhang, Y.; Jungreis, R.; Nagata, K.;
Harding, H.P.; Ron, D. CHOP induces death by promoting protein synthesis and oxidation in the
stressed endoplasmic reticulum. Genes Dev. 2004, 18, 3066–3077.
130. Tsaytler, P.; Harding, H.P.; Ron, D.; Bertolotti, A. Selective inhibition of a regulatory subunit of
protein phosphatase 1 restores proteostasis. Science 2011, 332, 91–94.
131. Calfon, M.; Zeng, H.; Urano, F.; Till, J.H.; Hubbard, S.R.; Harding, H.P.; Clark, S.G.; Ron, D.
IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA.
Nature 2002, 415, 92–96.
132. Yoshida, H.; Matsui, T.; Yamamoto, A.; Okada, T.; Mori, K. XBP1 mRNA is induced by ATF6
and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell
2001, 107, 881–891.
133. Shen, X.; Ellis, R.E.; Lee, K.; Liu, C.Y.; Yang, K.; Solomon, A.; Yoshida, H.; Morimoto, R.;
Kurnit, D.M.; Mori, K.; et al. Complementary signaling pathways regulate the unfolded protein
response and are required for C. elegans development. Cell 2001, 107, 893–903.
Int. J. Mol. Sci. 2015, 16 17223

134. Lee, K.; Tirasophon, W.; Shen, X.; Michalak, M.; Prywes, R.; Okada, T.; Yoshida, H.; Mori, K.;
Kaufman, R.J. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage
merge to regulate XBP1 in signaling the unfolded protein response. Genes Dev. 2002, 16,
452–466.
135. Yoshida, H.; Matsui, T.; Hosokawa, N.; Kaufman, R.J.; Nagata, K.; Mori, K. A time-dependent
phase shift in the mammalian unfolded protein response. Dev. Cell 2003, 4, 265–271.
136. Reimold, A.M.; Iwakoshi, N.N.; Manis, J.; Vallabhajosyula, P.; Szomolanyi-Tsuda, E.;
Gravallese, E.M.; Friend, D.; Grusby, M.J.; Alt, F.; Glimcher, L.H. Plasma cell differentiation
requires the transcription factor XBP-1. Nature 2001, 412, 300–307.
137. Korennykh, A.V.; Egea, P.F.; Korostelev, A.A.; Finer-Moore, J.; Stroud, R.M.; Zhang, C.;
Shokat, K.M.; Walter, P. Cofactor-mediated conformational control in the bifunctional
kinase/RNase Ire1. BMC Biol. 2011, 9, 48.
138. Brewer, J.W. Regulatory crosstalk within the mammalian unfolded protein response. Cell Mol.
Life Sci. 2014, 71, 1067–1079.
139. Hetz, C. The unfolded protein response: Controlling cell fate decisions under ER stress and
beyond. Nat. Rev. Mol. Cell Biol. 2012, 13, 89–102.
140. Lin, J.H.; Li, H.; Yasumura, D.; Cohen, H.R.; Zhang, C.; Panning, B.; Shokat, K.M.;
Lavail, M.M.; Walter, P. IRE1 signaling affects cell fate during the unfolded protein response.
Science 2007, 318, 944–949.
141. Li, H.; Korennykh, A.V.; Behrman, S.L.; Walter, P. Mammalian endoplasmic reticulum stress
sensor IRE1 signals by dynamic clustering. Proc. Natl. Acad. Sci. USA 2010, 107, 16113–16118.
142. Escusa-Toret S.; Vonk, W.I.; Frydman, J. Spatial sequestration of misfolded proteins by a
dynamic chaperone pathway enhances cellular fitness during stress. Nat. Cell Biol. 2013, 15,
1231–1243.
143. Duttler, S.; Pechmann, S.; Frydman, J. Principles of cotranslational ubiquitination and quality
control at the ribosome. Mol. Cell 2013, 50, 379–393.
144. Wang, F.; Durfee, L.A.; Huibregtse, J.M. A cotranslational ubiquitination pathway for quality
control of misfolded proteins. Mol. Cell 2013, 50, 368–378.
145. Pechmann, S.; Chartron, J.W.; Frydman, J. Local slowdown of translation by nonoptimal codons
promotes nascent-chain recognition by SRP in vivo. Nat. Struct. Mol. Biol. 2014, 21, 1100–1105.
146. Wolff, S.; Weissman, J.S.; Dillin, A. Differential scales of protein quality control. Cell 2014,
157, 52–64.
147. Douglas, P.M.; Summers, D.W.; Cyr, D.M. Molecular chaperones antagonize proteotoxicity by
differentially modulating protein aggregation pathways. Prion 2009, 3, 51–58.
148. Cohen, E.; Bieschke, J.; Perciavalle, R.M.; Kelly, J.W.; Dillin, A. Opposing activities protect
against age-onset proteotoxicity. Science 2006, 313, 1604–1610.
149. Arrasate, M.; Mitra, S.; Schweitzer, E.S.; Segal, M.R.; Finkbeiner, S. Inclusion body formation
reduces levels of mutant huntingtin and the risk of neuronal death. Nature 2004, 431, 805–810.
150. Kaganovich, D.; Kopito, R.; Frydman, J. Misfolded proteins partition between two distinct
quality control compartments. Nature 2008, 454, 1088–1095.
Int. J. Mol. Sci. 2015, 16 17224

151. Ogrodnik, M.; Salmonowicz, H.; Brown, R.; Turkowska, J.; Średniawa, W.; Pattabiraman, S.;
Amen, T.; Abraham, A.C.; Eichler, N.; Lyakhovetsky, R.; et al. Dynamic JUNQ inclusion bodies
are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of
vimentin. Proc. Natl. Acad. Sci. USA 2014, 11, 8049–8054.
152. Polling, S.; Mok, Y.F.; Ramdzan, Y.M.; Turner, B.J.; Yerbury, J.J.; Hill, A.F.; Hatters, D.M.
Misfolded polyglutamine, polyalanine, and superoxide dismutase 1 aggregate via distinct
pathways in the cell. J. Biol. Chem. 2014, 289, 6669–6690.
153. Weisberg, S.J.; Lyakhovetsky, R.; Werdiger, A.-C.; Gitler, A.D.; Soen, Y.; Kaganovich, D.
Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their
toxicity. Proc. Natl. Acad. Sci. USA 2012, 109, 15811–15816.
154. Cohen, S.I.; Arosio, P.; Presto, J.; Kurudenkandy, F.R.; Biverstål, H.; Dolfe, L.; Dunning, C.;
Yang, X.; Frohm, B.; Vendruscolo, M.; et al. A molecular chaperone breaks the catalytic cycle
that generates toxic Aβ oligomers. Nat. Struct. Mol. Biol. 2015, 22, 207–213.
155. Wrighton, K.H. Protein folding: Misfolded proteins join the Q. Nat. Rev. Mol. Cell Biol. 2013,
14, 608.
156. Labbadia, J.; Morimoto, R.I. The biology of proteostasis in aging and disease. Annu. Rev. Biochem.
2015, doi:10.1146/annurev-biochem-060614-033955.
157. Voisine, C.; Pedersen, J.S.; Morimoto, R.I. Chaperone networks: Tipping the balance in protein
folding diseases. Neurobiol. Dis. 2010, 40, 12–20.
158. Fredrickson, E.K.; Gardner, R.G. Selective destruction of abnormal proteins by ubiquitin-mediated
protein quality control degradation. Semin. Cell Dev. Biol. 2012, 23, 530–537.
159. He, C.; Klionsky, D.J. Regulation mechanisms and signaling pathways of autophagy.
Annu. Rev. Genet. 2009, 43, 67–93.
160. Schmidt, M.; Finley, D. Regulation of proteasome activity in health and disease.
Biochim. Biophys. Acta 2014, 1843, 13–25.
161. Murata, S.; Yashiroda, H.; Tanaka, K. Molecular mechanisms of proteasome assembly. Nat. Rev.
Mol. Cell Biol. 2009, 10, 104–115.
162. Kusmierczyk, A.R.; Kunjappu, M.J.; Funakoshi, M.; Hochstrasser, M. A multimeric assembly
factor controls the formation of alternative 20S proteasomes. Nat. Struct. Mol. Biol. 2008, 15,
237–244.
163. Konstantinova, I.M.; Tsimokha, A.S.; Mittenberg, A.G.; Kwang, W.J. Role of proteasomes in
cellular regulation. In International Review of Cell and Molecular Biology; Academic Press: San
Diego, CA, USA, 2008; Volume 267, pp. 59–124.
164. Da Fonseca, P.C.A.; He, J.; Morris, E.P. Molecular model of the human 26S proteasome.
Mol. Cell 2012, 46, 54–66.
165. Rosenzweig, R.; Osmulski, P.A.; Gaczynska, M.; Glickman, M.H. The central unit within the
19S regulatory particle of the proteasome. Nat. Struct. Mol. Biol. 2008, 15, 573–580.
166. Kikuchi, J.; Iwafune, Y.; Akiyama, T.; Okayama, A.; Nakamura, H.; Arakawa, N.; Kimura, Y.;
Hirano, H. Co- and post-translational modifications of the 26S proteasome in yeast. Proteomics
2010, 10, 2769–2779.
167. Tanaka, K. The proteasome: Overview of structure and functions. Proc. Jpn. Acad. Ser. B 2009,
85, 12–36.
Int. J. Mol. Sci. 2015, 16 17225

168. Li, X.; Demartino, G.N. Variably modulated gating of the 26S proteasome by ATP and
polyubiquitin. Biochem. J. 2009, 421, 397–404.
169. Sledź, P.; Förster, F.; Baumeister, W.J. Allosteric effects in the regulation of 26S proteasome
activities. Mol. Biol. 2013, 425, 1415–1423.
170. Price, J.L.; Davis, P.B.; Morris, J.C.; White, D.L. The distribution of tangles, plaques and related
immunohistochemical markers in healthy aging and Alzheimer’s disease. Neurobiol. Aging 1991,
12, 295–312.
171. Masters, C.L.; Simms, G.; Weinman, N.A.; Multhaup, G.; McDonald, B.L.; Beyreuther, K.
Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc. Natl. Acad.
Sci. USA 198, 82, 4245–4259.
172. Gregori, L.; Hainfeld, J.F.; Simon, M.N.; Goldgaber, D. Binding of amyloid β protein to the 20 S
proteasome. J. Biol. Chem. 1997, 272, 58–62.
173. Gregori, L.; Fuchs, C.; Figueiredo-Pereira, M.E.; van Nostrand, W.E.; Goldgaber, D. Amyloid
β-protein inhibits ubiquitin-dependent protein degradation in vitro. J. Biol. Chem. 1995, 270,
19702–19708.
174. Lopez Salon, M.; Pasquini, L.; Besio Moreno, M.; Pasquini, J.M.; Soto, E. Relationship between
β-amyloid degradation and the 26S proteasome in neural cells. Exp. Neurol. 2003, 180,
131–143.
175. Oh, S.; Hong, H.S.; Hwang, E.; Sim, H.J.; Lee, W.; Shin, S.J.; Mook-Jung, I. Amyloid peptide
attenuates the proteasome activity in neuronal cells. Mech. Ageing Dev. 2005, 126, 1292–1299.
176. Choi, A.M.; Ryter, S.W.; Levine, B. Autophagy in human health and disease. N. Engl. J. Med.
2013, 368, 651–662.
177. Wang, Z.V.; Hill, J.A. Protein quality control and metabolism: Bidirectional control in the heart.
Cell Metab. 2015, 21, 215–226.
178. Reggiori, F.; Klionsky, D.J. Autophagic processes in yeast: Mechanism, machinery and
regulation. Genetics 2013, 194, 341–361.
179. Noda, N.N.; Inagaki, F. Mechanisms of autophagy. Annu. Rev. Biophys. 2015, doi:10.1146/
annurev-biophys-060414-034248.
180. Cuervo, A.M.; Wong, E. Chaperone-mediated autophagy: Roles in disease and aging. Cell Res.
2014, 24, 92–104.
181. Yang, Z.; Klionsky, D.J. Mammalian autophagy: Core molecular machinery and signaling
regulation. Curr. Opin. Cell Biol. 2010, 22, 124–131.
182. Kroemer, G.; Mariño, G.; Levine, B. Autophagy and the integrated stress response. Mol. Cell
2010, 40, 280–293.
183. Levine, B.; Kroemer, G. Autophagy in the pathogenesis of disease. Cell 2008, 132, 27–42.
184. Yu, W.H.; Cuervo, A.M.; Kumar, A.; Peterhoff, C.M.; Schmidt, S.D.; Lee, J.H.; Mohan, P.S.;
Mercken, M.; Farmery, M.R.; Tjernberg, L.O.; et al. Macroautophagy a novel β-amyloid
peptide-generating pathway activated in Alzheimer’s disease. J. Cell Biol. 2005, 171, 87–98.
185. Rubinsztein, D.C.; Gestwicki, J.E.; Murphy, L.O.; Klionsky, D.J. Potential therapeutic applications
of autophagy. Nat. Rev. Drug Discov. 2007, 6, 304–312.
186. Ferraro, E.; Cecconi, F. Autophagic and apoptotic response to stress signals in mammalian cells.
Arch. Biochem. Biophys. 2007, 462, 210–219.
Int. J. Mol. Sci. 2015, 16 17226

187. Gavrin, L.K.; Denny, R.A.; Saiah, E. Small molecules that target protein misfolding. J. Med. Chem.
2012, 55, 10823–10843.
188. Ciryam, P.; Tartaglia, G.G.; Morimoto, R.I.; Dobson, C.M.; Vendruscolo, M. Widespread
aggregation and neurodegenerative diseases are associated with supersaturated proteins. Cell Rep.
2013, 5, 781–790.
189. Brocchieri, L.; Karlin, S. Protein length in eukaryotic and prokaryotic proteomes. Nucleic Acids Res.
2005, 33, 3390–3400.
190. Ogle, J.M.; Ramakrishnan, V. Structural insights into translational fidelity. Annu. Rev. Biochem.
2005, 74, 129–177.
191. Pechmann, S.; Willmund, F.; Frydman, J. The ribosome as a hub for protein quality control.
Mol. Cell 2013, 49, 411–421.
192. Huang, C.J.; Lin, C.Y.; Haataja, L.; Gurlo, T.; Butler, A.E.; Rizza, R.A.; Butler, P.C. High
expression rates of human islet amyloid polypeptide induce endoplasmic reticulum stress
mediated β-cell apoptosis, a characteristic of humans with type 2 but not type 1 diabetes.
Diabetes 2007, 56, 2016–2027.
193. Han, J.; Back, S.H.; Hur, J.; Lin, Y.H.; Gildersleeve, R.; Shan, J.; Yuan, C.L.; Krokowski, D.;
Wang, S.; Hatzoglou, M.; et al. ER-stress-induced transcriptional regulation increases protein
synthesis leading to cell death. Nat. Cell Biol. 2013, 15, 481–490.
194. Tyedmers, J.; Mogk, A.; Bukau, B. Cellular strategies for controlling protein aggregation.
Nat. Rev. Mol. Cell Biol. 2010, 11, 777–788.
195. Bence, N.F.; Sampat, R.M.; Kopito, R.R. Impairment of the ubiquitin-proteasome system by
protein aggregation. Science 2001, 292, 1552–1555.
196. Ross, C.A.; Poirier, M.A. Protein aggregation and neurodegenerative disease. Nat. Med. 2004,
10, S10–S17.
197. Glickman, M.H.; Ciechanover, A. The ubiquitin-proteasome proteolytic pathway: Destruction for
the sake of construction. Physiol. Rev. 2002, 82, 373–428.
198. Rubinsztein, D.C. The roles of intracellular protein-degradation pathways in neurodegeneration.
Nature 2006, 443, 780–786.
199. Mizushima, N.; Levine, B.; Cuervo, A.M.; Klionsky, D.J. Autophagy fights disease through
cellular self-digestion. Nature 2008, 451, 1069–1075.
200. Knowles, T.P.J.; Shu, W.; Devlin, G.L.; Meehan, S.; Auer, S.; Dobson, C.M.; Welland, M.E.
Kinetics and thermodynamics of amyloid formation from direct measurements of fluctuations in
fibril mass. Proc. Natl. Acad. Sci. USA 2007, 104, 10016–10021.
201. Xu, L.Q.; Wu, S.; Buell, A.K.; Cohen, S.I.; Chen, L.J.; Hu, W.H.; Cusack, S.A.; Itzhaki, L.S.;
Zhang, H.; Knowles, T.P.; et al. Influence of specific Hsp70 domains on fibril formation of the
yeast prion protein Ure2. Philos. Trans. R. Soc. B 2013, 368, 20110410.
202. Lee, J.; Ozcan, U. Unfolded protein response signaling and metabolic diseases. J. Biol. Chem.
2014, 289, 1203–1211
203. Huang, A.; Stultz, C.M. Finding order within disorder: Elucidating the structure of proteins
associated with neurodegenerative disease. Future Med. Chem. 2009, 1, 467–482.
204. Cnop, M.; Igoillo-Esteve, M.; Cunha, D.A.; Ladrière, L.; Eizirik, D.L. An update on lipotoxic
endoplasmic reticulum stress in pancreatic β-cells. Biochem. Soc. Trans. 2008, 36, 909–915.
Int. J. Mol. Sci. 2015, 16 17227

205. Hetz, C.; Mollereau, B. Disturbance of endoplasmic reticulum proteostasis in neurodegenerative

diseases. Nat. Rev. Neurosci. 2014, 15, 233–249.
206. Steiner, H.; Haass, C. Intramembrane proteolysis by presenilins. Nat. Rev. Mol. Cell Biol. 2000,
1, 217–224.
207. Van Laar, V.S.; Arnold, B.; Cassady, S.J.; Chu, C.T.; Burton, E.A.; Berman, S.B.; Bioenergetics
of neurons inhibit the translocation response of Parkin following rapid mitochondrial
depolarization. Hum. Mol. Genet. 2011, 20, 927–940.
208. Cao, S.S.; Kaufman, R.J. Targeting endoplasmic reticulum stress in metabolic disease. Expert Opin.
Ther. Targets 2012, 17, 437–448.
209. Hotamisligil, G.S. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease.
Cell 2010, 140, 900–917.
210. Cao, P.; Abedini, A.; Raleigh, D.P. Aggregation of islet amyloid polypeptide: From physical
chemistry to cell biology. Curr. Opin. Struct. Biol. 2013, 23, 82–89.
211. Fernández, M.S. Human IAPP amyloidogenic properties and pancreatic β-cell death. Cell Calcium
2014, 56, 416–427.
212. Casas, S.; Gomis, R.; Gribble, F.M.; Altirriba, J.; Knuutila, S.; Novials, A. Impairment of the
ubiquitin-proteasome pathway is a downstream endoplasmic reticulum stress response induced
by extracellular human islet amyloid polypeptide and contributes to pancreatic β-cell apoptosis.
Diabetes 2007, 56, 2284–2294.
213. Costes, S.; Huang, C.J.; Gurlo, T.; Daval, M.; Matveyenko, A.V.; Rizza, R.A.; Butler, A.E.;
Butler, P.C. β-cell dysfunctional ERAD/ubiquitin/proteasome system in type 2 diabetes mediated
by islet amyloid polypeptide-induced UCH-L1 deficiency. Diabetes 2011, 60, 227–238.
214. Ozcan, U.; Cao, Q.; Yilmaz, E.; Lee, A.H.; Iwakoshi, N.N.; Ozdelen, E.; Tuncman, G.; Görgün, C.;
Glimcher, L.H.; Hotamisligil, G.S. Endoplasmic reticulum stress links obesity, insulin action,
and type 2 diabetes. Science 2004, 306, 457–461.
215. Welters, H.J.; Tadayyon, M.; Scarpello, J.H.; Smith, S.A.; Morgan, N.G. Mono-unsaturated fatty
acids protect against β-cell apoptosis induced by saturated fatty acids, serum withdrawal or
cytokine exposure. FEBS Lett. 2004, 560, 103–108.
216. Díaz-Villanueva, J.F.; García-González, V. Key interactions in the binding of free fatty acids on
amylin-structure transitions. 2015, manuscript in preparation.
217. Tabas, I. The role of endoplasmic reticulum stress in the progression of atherosclerosis. Circ. Res.
2010, 107, 839–850.
218. Kaufman, R.J. Orchestrating the unfolded protein response in health and disease. J. Clin. Investig.
2002, 110, 1389–1398.
219. Han, D.; Lerner, A.G.; Vande, W.L.; Upton, J.P.; Xu, W.; Hagen, A.; Backes, B.J.; Oakes, S.A.;
Papa, F.R. IRE1α kinase activation modes control alternate endoribonuclease outputs to
determine divergent cell fates. Cell 2009, 138, 562–575.
220. Blais, J.D.; Chin, K.T.; Zito, E.; Zhang, Y.; Heldman, N.; Harding, H.P.; Fass, D.; Thorpe, C.;
Ron, D.A. Small molecule inhibitor of endoplasmic reticulum oxidation 1 (ERO1) with
selectively reversible thiol reactivity. J. Biol. Chem. 2010, 285, 20993–21003.
221. Ma, Y.; Hendershot, L.M. The role of the unfolded protein response in tumour development:
Friend or foe? Nat. Rev. Cancer 2004, 4, 966–977.
Int. J. Mol. Sci. 2015, 16 17228

222. Moenner, M.; Pluquet, O.; Bouchecareilh, M.; Chevet, E. Integrated endoplasmic reticulum
stress responses in cancer. Cancer Res. 2007, 67, 10631–10634.
223. Rouschop, K.M.; van den Beucken, T.; Dubois, L.; Niessen, H.; Bussink, J.; Savelkouls, K.;
Keulers, T.; Mujcic, H.; Landuyt, W.; Voncken, J.W.; et al. The unfolded protein response
protects human tumor cells during hypoxia through regulation of the autophagy genes
MAP1LC3B and ATG5. J. Clin. Investig. 2010, 120, 127–141.
224. Blais, J.D.; Addison, C.L.; Edge, R.; Falls, T.; Zhao, H.; Wary, K.; Koumenis, C.; Harding, H.P.;
Ron, D.; Holcik, M.; et al. Perk-dependent translational regulation promotes tumor cell
adaptation and angiogenesis in response to hypoxic stress. Mol. Cell. Biol. 2006, 26, 9517–9532.
225. Spiotto, M.T.; Banh, A.; Papandreou, I.; Cao, H.; Galvez, M.G.; Gurtner, G.C.; Denko, N.C.;
Le, Q.T.; Koong, A.C. Imaging the unfolded protein response in primary tumors reveals
microenvironments with metabolic variations that predict tumor growth. Cancer Res. 2010, 70,
78–88.
226. Lee, A.S. GRP78 induction in cancer: Therapeutic and prognostic implications. Cancer Res.
2007, 67, 3496–3499.
227. Pyrko, P.; Schonthal, A.H.; Hofman, F.M.; Chen, T.C.; Lee, A.S. The unfolded protein response
regulator GRP78/BiP as a novel target for increasing chemosensitivity in malignant gliomas.
Cancer Res. 2007, 67, 9809–9816.
228. Chen, T.C. GRP78/BiP modulation of GRP78/BiP in altering sensitivity to chemotherapy.
Methods Enzymol. 2011, 491, 25–36.
229. Li, X.; Zhang, K.; Li, Z. Unfolded protein response in cancer: The physician’s perspective.
J. Hematol. Oncol. 2011, 4, 8.
230. Park, H.R.; Tomida, A.; Sato, S.; Tsukumo, Y.; Yun, J.; Yamori, T.; Hayakawa, Y.; Tsuruo, T.;
Shin-ya, K. Effect on tumor cells of blocking survival response to glucose deprivation. J. Natl.
Cancer Inst. 2004, 96, 1300–1310.
231. Saito, S.; Furuno, A.; Sakurai, J.; Sakamoto, A.; Park, H.R.; Shin-Ya, K.; Tsuruo, T.; Tomida, A.
Chemical genomics identifies the unfolded protein response as a target for selective cancer cell
killing during glucose deprivation. Cancer Res. 2009, 69, 4225–4234.
232. Backer, J.M.; Krivoshein, A.V.; Hamby, C.V.; Pizzonia, J.; Gilbert, K.S.; Ray, Y.S.; Brand, H.;
Paton, A.W.; Paton, J.C.; Backer, M.V. Chaperone-targeting cytotoxin and endoplasmic
reticulum stress-inducing drug synergize to kill cancer cells. Neoplasia 2009, 11, 1165–1173.
233. Brown, C.J.; Lain, S.; Verma, C.S.; Fersht, A.R.; Lane, D.P. Awakening guardian angels:
Drugging the p53 pathway. Nat. Rev. Cancer 2009, 12, 862–873.
234. Olivier, M.; Hollstein, M.; Hainaut, P. TP53 mutations in human cancers: Origins, consequences,
and clinical use. Cold Spring Harb. Perspect. Biol. 2010, 1, a001008.
235. Cho, Y.; Gorina, S.; Jeffrey, P.D.; Pavletich, N.P. Crystal structure of a p53 tumor
suppressor-DNA complex: Understanding tumorigenic mutations. Science 1994, 265, 346–355.
236. Segalat, L. Loss-of-function genetic diseases and the concept of pharmaceutical targets.
Orphanet. J. Rare Dis. 2007, 2, 30–36.
237. Rippin, T.M.; Bykov, V.J.; Freund, S.M.; Selivanova, G.; Wiman, K.G.; Fersht, A.R.
Characterization of the p53-rescue drug CP-31398 in vitro and in living cells. Oncogene 2002,
21, 2119–2129.
Int. J. Mol. Sci. 2015, 16 17229

238. Friedler, A.; Hansson, L.O.; Veprintsev, D.B.; Freund, S.M.; Rippin, T.M.; Nikolova, P.V.;
Proctor, M.R.; Rüdiger, S.; Fersht, A.R. A peptide that binds and stabilizes p53 core domain:
Chaperone strategy for rescue of oncogenic mutants. Proc. Natl. Acad. Sci. USA 2002, 99,
937–942.
239. Ellis, R.J. Macromolecular crowding: An important but neglected aspect of the intracellular
environment. Curr. Opin. Struct. Biol. 2001, 11, 114–119.
240. Gribbin, J.R. Deep Simplicity: Bringing Order to Chaos and Complexity, 1st ed.; Random House
Inc.: New York, NY., USA, 2004; pp. 1–304.
241. Baigent, C.; Keech, A.; Kearney, P.M.; Blackwell, L.; Buck, G.; Pollicino, C.; Kirby, A.;
Sourjina, T.; Peto, R.; Collins, R.; et al. Efficacy and safety of cholesterol lowering treatment:
Prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins.
Lancet 2005, 366, 1267–1278.
242. Perutz, M.F.; Pope, B.J.; Owen, D.; Wanker, E.E.; Scherzinger, E. Aggregation of proteins with
expanded glutamine and alanine repeats of the glutamine-rich and asparagine rich domains of
Sup35 and of the amyloid β-peptide of amyloid plaques. Proc. Natl. Acad. Sci. USA 2002, 99,
5596–5600.
243. Guijarro, J.I.; Sunde, M.; Jones, J.A.; Campbell, I.D.; Dobson. C.M. Amyloid fibril formation by
an SH3 domain. Proc. Natl. Acad. Sci. USA 1998, 95, 4224–4228.
244. Wasmer, C.; Schütz, A.; Loquet, A.; Buhtz, C.; Greenwald, J.; Riek, R.; Böckmann, A.; Meier, B.H.
The molecular organization of the fungal prion HET-s in its amyloid form. J. Mol. Biol. 2009,
394, 119–127.
245. Johnson, S.M.; Wiseman, R.L.; Sekijima, Y.; Green, N.S.; Adamski-Werner, S.L.; Kelly, J.W.
Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: A focus
on the transthyretin amyloidoses. Acc. Chem. Res. 2005, 38, 911–921.
246. Lansbury, P. Evolution of amyloid: What normal protein folding may tell us about fibrillogenesis
and disease? Proc. Natl. Acad. Sci. USA 1999, 96, 3342–3344.
247. Hetz, C.; Chevet, E.; Harding, H.P. Targeting the unfolded protein response in disease. Nat. Rev.
Drug Discov. 2013, 12, 703–719.
248. Kraskiewicz, H.; FitzGerald, U. InterfERing with endoplasmic reticulum stress. Trends Pharmacol. Sci.
2012, 33, 53–63.
249. Lindquist, S.L.; Kelly, J.W. Chemical and biological approaches for adapting proteostasis to
ameliorate protein misfolding and aggregation diseases: Progress and prognosis. Cold Spring
Harb. Perspect. Biol. 2011, 3, a004507.
250. Ozcan, U.; Yilmaz, E.; Ozcan, L.; Furuhashi, M.; Vaillancourt, E.; Smith, R.O.; Görgün, C.Z.;
Hotamisligil, G.S. Chemical chaperones reduce ER stress and restore glucose homeostasis in a
mouse model of type 2 diabetes. Science 2006, 313, 1137–1140.
251. Ozcan, L.; Ergin, A.S.; Lu, A.; Chung, J.; Sarkar, S.; Nie, D.; Myers, M.G., Jr.; Ozcan, U.
Endoplasmic reticulum stress plays a central role in development of leptin resistance. Cell Metab.
2009, 9, 35–51.
252. Xiao, C.; Giacca, A.; Lewis, G.F. Sodium phenylbutyrate, a drug with known capacity to reduce
endoplasmic reticulum stress, partially alleviates lipid-induced insulin resistance and β-cell
dysfunction in humans. Diabetes 2011, 60, 918–924.
Int. J. Mol. Sci. 2015, 16 17230

253. Kars, M.; Yang, L.; Gregor, M.F.; Mohammed, B.S.; Pietka, T.A.; Finck, B.N.; Patterson, B.W.;
Horton, J.D.; Mittendorfer, B.; Hotamisligil, G.S.; et al. Tauroursodeoxycholic Acid may
improve liver and muscle but not adipose tissue insulin sensitivity in obese men and women.
Diabetes 2010, 59, 1899–1905
254. Promlek, T.; Ishiwata-Kimata, Y.; Shido, M.; Sakuramoto, M.; Kohno, K.; Kimata, Y.
Membrane aberrancy and unfolded proteins activate the endoplasmic reticulum stress sensor Ire1
in different ways. Mol. Biol. Cell 2011, 22, 3520–3532.
255. Wiseman, R.L.; Zhang, Y.; Lee, K.P.; Harding, H.P.; Haynes, C.M.; Price, J.; Sicheri, F.;
Ron, D. Flavonol activation defines an unanticipated ligand-binding site in the kinase-RNase
domain of IRE1. Mol. Cell 2010, 38, 291–304.
256. Foster, B.A.; Coffey, H.A.; Morin, M.J.; Rastinejad, F. Pharmacological rescue of mutant p53
conformation and function. Science 1999, 286, 2507–2510.

© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).